Microfluidic cartridge

ABSTRACT

The technology described herein generally relates to microfluidic cartridges configured to amplify and detect polynucleotides extracted from multiple biological samples in parallel. The technology includes a microfluidic substrate, comprising: a plurality of sample lanes, wherein each of the plurality of sample lanes comprises a microfluidic network having, in fluid communication with one another: an inlet; a first valve and a second valve; a first channel leading from the inlet, via the first valve, to a reaction chamber; and a second channel leading from the reaction chamber, via the second valve, to a vent.

CLAIM OF PRIORITY

This application is a continuation-in-part of U.S. patent applicationSer. No. 12/173,023 and U.S. patent application Ser. No. 12/218,498,both filed on Jul. 14, 2008, which claim benefit of priority to U.S.provisional patent application Ser. No. 60/959,437, filed Jul. 13, 2007all of which are incorporated herein by reference in their entirety.This application is also a continuation-in-part of U.S. utilityapplication Ser. No. 11/985,577, filed Nov. 14, 2007, through whichbenefit of priority is claimed to U.S. provisional patent applicationSer. No. 60/859,284, filed Nov. 14, 2006, both of which are incorporatedherein by reference in their entirety. This application also claimsbenefit of priority to U.S. design patent application Ser. No.29/308,920, filed Jul. 14, 2008, and incorporated herein by reference inits entirety.

TECHNICAL FIELD

The technology described herein generally relates to microfluidiccartridges. The technology more particularly relates to microfluidiccartridges that are configured to carry out PCR on nucleotides ofinterest, particularly from multiple biological samples in parallel,within microfluidic channels, and permit detection of those nucleotides.

BACKGROUND

The medical diagnostics industry is a critical element of today'shealthcare infrastructure. At present, however, diagnostic analyses nomatter how routine have become a bottleneck in patient care. There areseveral reasons for this. First, many diagnostic analyses can only bedone with highly specialist equipment that is both expensive and onlyoperable by trained clinicians. Such equipment is found in only a fewlocations—often just one in any given urban area. This means that mosthospitals are required to send out samples for analyses to theselocations, thereby incurring shipping costs and transportation delays,and possibly even sample loss or mishandling. Second, the equipment inquestion is typically not available ‘on-demand’ but instead runs inbatches, thereby delaying the processing time for many samples becausethey must wait for a machine to fill up before they can be run.

Understanding that sample flow breaks down into several key steps, itwould be desirable to consider ways to automate as many of these aspossible. For example, a biological sample, once extracted from apatient, must be put in a form suitable for a processing regime thattypically involves using PCR to amplify a vector of interest. Onceamplified, the presence or absence of a nucleotide of interest from thesample needs to be determined unambiguously. Preparing samples for PCRis currently a time-consuming and labor intensive step, though not onerequiring specialist skills, and could usefully be automated. Bycontrast, steps such as PCR and nucleotide detection have customarilyonly been within the compass of specially trained individuals havingaccess to specialist equipment.

There is a need for a method and apparatus of carrying out samplepreparation on samples in parallel, followed by PCR and detection on theprepared biological samples, and preferably with high throughput. ThePCR should be capable of being carried out by someone requiring minimaltraining, and should be fast.

The discussion of the background herein is included to explain thecontext of the inventions described herein. This is not to be taken asan admission that any of the material referred to was published, known,or part of the common general knowledge as at the priority date of anyof the claims.

Throughout the description and claims of the specification the word“comprise” and variations thereof, such as “comprising” and “comprises”,is not intended to exclude other additives, components, integers orsteps.

SUMMARY

The present technology includes a microfluidic substrate, comprising: aplurality of sample lanes, wherein each of the plurality of sample lanescomprises a microfluidic network having, in fluid communication with oneanother: an inlet; a first valve and a second valve; a first channelleading from the inlet, via the first valve, to a reaction chamber; anda second channel leading from the reaction chamber, via the secondvalve, to a vent. The present technology further includes a microfluidiccartridge comprising a microfluidic substrate having the aforementionedfeatures.

The technology further includes a microfluidic cartridge, consisting of:a substrate having an upper side and an opposed lower side, wherein thesubstrate comprises a plurality of microfluidic networks arranged into aplurality of sample lanes; a laminate attached to the lower side; and alabel, attached to the upper side. The cartridge can be furtherconfigured such that each lane of the plurality of lanes comprises amicrofluidic network having, in fluid communication with one another: aninlet; a first valve and a second valve; a first channel leading fromthe inlet, via the first valve, to a reaction chamber; and a secondchannel leading from the reaction chamber, via the second valve, to avent.

The technology further includes a method of carrying out PCRindependently on a plurality of polynucleotide-containing samples, themethod comprising: introducing the plurality of samples into amicrofluidic cartridge, wherein the cartridge has a plurality of PCRreaction chambers configured to permit thermal cycling of the pluralityof samples independently of one another; moving the plurality of samplesindependently of one another into the respective plurality of PCRreaction chambers; isolating the plurality of PCR reaction chambers; andamplifying polynucleotides contained with the plurality of samples, byapplication of successive heating and cooling cycles independently tothe PCR reaction chambers. In certain implementations, PCR is carriedout simultaneously on two or more of the plurality of samples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exemplary microfluidic cartridge having a 3-layerconstruction.

FIGS. 2A-2E show various views of an embodiment of a microfluidiccartridge.

FIG. 3 shows a perspective view of an embodiment of a microfluidiccartridge.

FIGS. 4A-4D show various views of an embodiment of a microfluidiccartridge.

FIG. 4E shows an overlay of a heater unit with the lanes of thecartridge.

FIGS. 5A-5B show diagrams of an exemplary microfluidic double valve.FIG. 5A additionally shows an exemplary valve in an open state, and thevalve in a closed state.

FIGS. 6A-6B show diagrams of exemplary single microfluidic valves.

FIG. 7 shows a hydrophobic vent suitable for use in a microfluidicnetwork described elsewhere.

FIG. 8 shows an assembly process for a cartridge as further describedherein.

FIGS. 9A and 9B show exemplary deposition of wax droplets intomicrofluidic valves.

FIGS. 10A and 10B show an exemplary apparatus for carrying out waxdeposition.

FIG. 11 shows a schematic of a diagnostic apparatus.

FIGS. 12A and 12B show exterior views of an exemplary apparatus.

FIG. 13 shows an exemplary interior view of an apparatus, illustrating acartridge receiving bay.

FIG. 14 shows a microfluidic cartridge, and a cartridge tray.

FIG. 15 shows a cross-section of a pipetting head and a cartridge inposition in a microfluidic apparatus.

FIG. 16 shows a view in cross-section of a microfluidic cartridgesituated in a receiving bay, adjacent a heater unit.

FIGS. 17A, 17B show a PCR reaction chamber and associated heaters.

FIG. 18 shows thermal images of heater circuitry in operation.

FIGS. 19A-19C shows various cut-away sections that can be used toimprove cooling rates during PCR thermal cycling.

FIG. 20 shows a plot of temperature against time during a PCR process,as performed on a microfluidic cartridge as described herein.

FIGS. 21A-21C show views of an exemplary highly-multiplexed microfluidiccartridge, in plan (FIG. 21A), close-up of valves (FIG. 21B), and closeup of inlets (FIG. 21C).

FIGS. 22A and 22B show various aspects of heater units associated withexemplary highly multiplexed microfluidic cartridges.

FIGS. 23A-C show various aspects of a radially configured highlymultiplexed microfluidic cartridge.

FIG. 24 shows a cartridge auto-loader in conjunction with anamplification-detection system.

FIG. 25 shows a perspective view, and a close-up view, of a cartridgestacker.

FIG. 26 shows a cartridge stacker in position to deliver a cartridge toan auto-loader.

FIG. 27 shows a cartridge loading system.

FIG. 28 shows a disposal unit for used cartridges.

FIG. 29 shows a cartridge stacker in full and empty configurations.

FIG. 30 shows a barcode reader positioned above a microfluidiccartridge.

FIGS. 31A and 31B show a thermocycling unit configured to accept amicrofluidic cartridge.

FIG. 32A shows a plan view of a 24-lane microfluidic cartridge.

FIG. 32B shows a perspective view of the cartridge of FIG. 32A.

FIG. 32C shows an exploded view of the cartridge of FIG. 32A.

DETAILED DESCRIPTION

The present technology includes microfluidic cartridges that areconfigured to carry out PCR on multiple PCR-ready nucleic acidcontaining samples. The PCR-ready samples have typically been preparedby other devices, or components of an apparatus that accommodates themicrofluidic cartridge during use, and introduced into the microfluidiccartridge prior to initiation of PCR.

The microfluidic cartridges described herein are particularly effectivefor high throughput PCR applications because, for example, the smallvolumes of sample that are involved permit rapid heating and coolingcycles, and also mean that the PCR can be carried out on all of thesamples in parallel in a relatively small space, thereby facilitatingreal-time analysis of multiple samples on a benchtop in a clinicalsetting.

Nucleic acid testing (NAT) as used herein is a general term thatencompasses both DNA (deoxyribonucleic acid) and RNA (ribonucleic acid)testing. Exemplary protocols that are specific to RNA and to DNA aredescribed herein. It is to be understood that generalized descriptionswhere not specific to RNA or to DNA either apply to each equally or canbe readily adapted to either with minor variations of the descriptionherein as amenable to one of ordinary skill in the art. The termsnucleic acid and polynucleotide are used interchangeably herein.

Where used herein, the term “module” should be taken to mean an assemblyof components, each of which may have separate, distinct and/orindependent functions, but which are configured to operate together toproduce a desired result or results. It is not required that everycomponent within a module be directly connected or in directcommunication with every other. Furthermore, connectivity amongst thevarious components may be achieved with the aid of a component, such asa processor, that is external to the module.

The microfluidic cartridges described herein are typically configuredfor use in a method and apparatus for carrying out sample preparation onbiological samples in parallel, to create prepared (PCR-ready) samples,with PCR and detection on the prepared samples, and preferably with highthroughput. Aspects of the operation of such apparatuses and theirmanner of communicating with microfluidic cartridges, are furtherdescribed herein

Microfluidic Cartridge

One aspect of the present technology relates to a microfluidic cartridgethat includes a first, second, and third layers that together define aplurality of microfluidic networks, each network having variouscomponents configured to carry out PCR on a sample having one or morepolynucleotides whose presence is to be determined. The cartridgeincludes one or more sample lanes in parallel, wherein each lane isindependently associated with a given sample for simultaneousprocessing, and each lane contains an independently configuredmicrofluidic network. The cartridge typically processes the one or moresamples by increasing the concentration of (such as by amplification)one or more polynucleotides to be determined, as present in each of thesamples. An exemplary cartridge having such a construction is shown inFIG. 1. Such a cartridge is simple to manufacture, and permits PCR to becarried out in a concentrated reaction volume (˜4 μl) and enables rapidthermocycling, at ˜20 seconds per cycle.

Although other layers may be found in cartridges having comparableperformance and ease of manufacture, the cartridge herein includesembodiments having only three layers in their construction, as shown inthe embodiment 3000 of FIG. 1, which has a substrate 3004, a laminate3006, and a label 3002. In cartridge 3000, a microfluidic substrate 3004has an upper side 3005 and, on an opposite side of the substrate (notvisible in FIG. 1), a lower side 3007, wherein the substrate comprises aplurality of microfluidic networks, arranged into a correspondingplurality of sample lanes 3003; a laminate 3006 attached to the lowerside 3007 of the substrate to seal the various components (such asvalves) of the microfluidic networks, and to provide an effectivethermal transfer layer between a dedicated heating element (furtherdescribed herein) and components in the microfluidic networks; and alabel 3002, attached to the upper side 3005 of the substrate 3004, whichalso covers and seals holes that are used in the manufacturing processto load components such as valves of the microfluidic networks withthermally responsive materials.

Thus, embodiments of microfluidic cartridges herein include embodimentsconsisting of three layers, a substrate, a laminate, and a label, thoughother, additional, features other than layers may be consistent withsuch characterizations. Embodiments herein further include microfluidiccartridges consisting essentially of three layers, a substrate, alaminate, and a label, though other, additional, features other thanlayers may be consistent with such characterizations. Furthermore,embodiments herein still further include microfluidic cartridgescomprising three layers, a substrate, a laminate, and a label.

The microfluidic substrate layer 3004 is typically injection molded outof a plastic, preferably a zeonor plastic (cyclic olefin polymer), andcontains a number of microfluidic networks (not shown in FIG. 1), eachhaving a PCR chamber, channels, and valves on a first (e.g., lower) side(disposed towards the laminate), and vent channels and various inletholes, including wax loading holes and liquid inlet holes, on a second(e.g., upper) side (disposed toward the label). Typically, in a givencartridge, all of the microfluidic networks together, including the PCRreactors, the inlet holes and the valves for isolating the PCR reactionchambers, are defined in a single substrate 3004. The substrate is madeof a material that confers rigidity on the substrate (and hence thecartridge), and is impervious to air or liquid, so that entry or exit ofair or liquid during operation of the cartridge is only possible throughthe inlets or the various vent.

Channels of a microfluidic network in a lane of cartridge 3000 typicallyhave at least one sub-millimeter cross-sectional dimension. For example,channels of such a network may have a width and/or a depth of less than1 mm (e.g., about 750 microns or less, about 500 microns, or less, orabout 250 microns or less).

The heat sealable laminate layer 3006 (typically between about 100 andabout 125 microns thick) can be attached to the bottom surface of themicrofluidic substrate using, for example, heat bonding, pressurebonding, or a combination thereof. The laminate layer 3006 may also bemade from a material that has an adhesive coating on one side only, thatside being the side that contacts the underside of the microfluidicsubstrate. This layer may be made from a single coated tape having alayer of Adhesive 420, made by 3M. Exemplary tapes include single-sidedvariants of double sided tapes having product nos. 9783, 9795, and9795B, and available from 3M. The laminate layer is typically 50-200μthick, for example 125μ thick. Other acceptable layers may be made fromadhesive tapes that utilize micro-capsule based adhesives.

Typically, the label 3002 is made from polypropylene or other plasticwith pressure sensitive adhesive (typically between about 50 and 150microns thick) and is configured to seal the wax loading holes of thevalves in the substrate, trap air used for valve actuation, and serve asa location for operator markings. The label can be in two or moreseparate pieces, though it would be understood by one of ordinary skillin the art that in many embodiments a single piece layer would beappropriate.

The label may be printed with various information, such as amanufacturer's logo, a part number, index numbers for each of the samplelanes, and an area 3012 where a user can make annotations. In variousembodiments, the label comprises a computer-readable or scannableportion 3008 that may contain certain identifying indicia such as a lotnumber, expiry date, or a unique identifier. For example, the label caninclude a bar code, a radio frequency tag, or one or morecomputer-readable, or optically scannable, characters. The readableportion of the label can be positioned such that it can be read by asample identification verifier as further described herein.

Microfluidic cartridge 3000 can be fabricated as desired, as furtherdescribed herein.

A multi-lane cartridge, such as shown in FIG. 1, is configured to accepta number of samples, in particular embodiments 12 samples, through anumber of inlets 3013 wherein the samples include at least a firstsample and a second sample, wherein the first sample and the secondsample each contain one or more polynucleotides in a form suitable foramplification. The polynucleotides in question may be the same as, ordifferent from one another, in different lanes of a cartridge. Themulti-sample cartridge comprises at least a first microfluidic networkand a second microfluidic network, adjacent to one another, wherein eachof the first microfluidic network and the second microfluidic network isas elsewhere described herein, and wherein the first microfluidicnetwork accepts the first sample, and wherein the second microfluidicnetwork accepts the second sample.

A sample lane, as further described herein, includes a network ofmicrofluidic elements, controllable independently of those in anothersample lane, by which a sample can be accepted and analyzed, accordingto methods described herein. A lane comprises at least a sample inlet,and a microfluidic component, as further described elsewhere herein. Insome embodiments, each microfluidic network additionally comprises anoverflow reservoir to contain surplus liquid dispensed into thecartridge.

A microfluidic network, in a microfluidic substrate of a cartridge, caninclude, in fluidic communication, one or more components selected fromthe group consisting of: gates, valves such as thermally actuatedvalves, channels, vents, and reaction chambers. Particular components ofexemplary microfluidic networks are further described elsewhere herein.

The microfluidic network in each lane is typically configured to carryout PCR on a PCR-ready sample, such as one containing nucleic acid (DNAor RNA) extracted from a raw biological sample using other aspects ofthe apparatus as further described herein. A PCR-ready sample is thustypically a mixture comprising the PCR reagent(s) and the neutralizedpolynucleotide sample, suitable for subjecting to thermal cyclingconditions that create PCR amplicons from the neutralized polynucleotidesample. For example, a PCR-ready sample can include a PCR reagentmixture comprising a polymerase enzyme, a positive control plasmid, afluorogenic hybridization probe selective for at least a portion of theplasmid and a plurality of nucleotides, and at least one probe that isselective for a polynucleotide sequence. Examples of such reagents, andprobes, are described elsewhere herein. A sample lane further typicallyincludes a region 3014 of the substrate above a PCR reactor that permitsa detector to monitor progress of the reaction and also to detectfluorescence from a probe that binds to a quantity of amplifiednucleotides.

In various embodiments, a lane of a microfluidic cartridge can include asample inlet port 3013, a first thermally actuated valve, a secondthermally actuated valve, a PCR reaction chamber, and one or morechannels connecting the inlet port to the PCR reaction chamber via thefirst valve, and one or more channels connecting the PCR reactionchamber to an exit vent via the second valve. The sample inlet valve canbe configured to accept a quantity of sample at a pressure differentialcompared to ambient pressure of between about 100 to 5,000 Pa, such ascan be delivered by an automated liquid dispenser (as described in U.S.patent application Ser. No. 12/212,403, filed Sep. 17, 2008, andincorporated herein by reference in its entirety). It should be notedthat the lower the loading pressure, the higher the fill time for aaliquot of reaction mix to fill the microfluidic network. Applying morepressure will reduce the fill time, but if the time for which thepressure is applied is not determined correctly, the sample could beblown out through the microfluidic cartridge (if an end hydrophobic ventis not present). Therefore the time for which the pressure is appliedshould to be properly determined, such as by methods available to one ofordinary skill in the art, to prevent underfill or overfill. In general,the fill time is inversely proportional to the viscosity of thesolution.

The sample inlets of adjacent lanes are reasonably spaced apart from oneanother to prevent any contamination of one sample inlet from anothersample when a user introduces a sample into any one cartridge. In someembodiments, the multi-sample cartridge is designed so that a spacingbetween the centroids of sample inlets is 8 mm, which is anindustry-recognized standard. This means that, in certain embodimentsthe center-to-center distance between inlet holes in the cartridge, asfurther described herein, is 8 mm. A spacing of 8 mm is convenient bothfor interfacing the cartridge with an automated pipetting apparatus, asdescribed elsewhere herein, as well as for interfacing with pipettescontrolled manually that, e.g., transfer samples from PCR tubes to thecartridge. Thus, when used in conjunction with an automated sampleloader having 4 heads, spaced equidistantly at 8 mm apart, the inletshaving a 8 mm spacing can be loaded in three batches of 4 inlets: e.g.,inlets 1, 4, 7, and 10 together, followed by 2, 5, 8, and 11, thenfinally 3, 6, 9, and 12, wherein the 12 inlets are numberedconsecutively from one side of the cartridge to the other.

One skilled in the art would recognize that other spacings, such as 6mm, 9 mm, 10 mm, and 12 mm, between centroids of sample inlets areconsistent operation of the cartridge, as described elsewhere herein.

In some embodiments, the sample inlets are configured so as to preventsubsequent inadvertent introduction of sample into a given lane after asample has already been introduced into that lane.

The inlet holes 3013 can be manufactured frusto-conical in shape with anappropriate conical angle so that industry-standard pipette tips (2 μl,20 μl, 200 μl, volumes, etc.) fit snugly, entering from the widest pointof the inlet. Thus, in certain embodiments, an inlet comprises aninverted frustoconical structure of at least 1 mm height, and having adiameter at its widest point that accepts entry of a pipette tip, offrom 1-5 mm. The apparatus herein may be adapted to suit other,later-arising, industry standards for pipette tips not otherwisedescribed herein. Typically the volume of sample accepted via an inletinto a microfluidic network in a sample lane is from 1-20 μl, and may befrom 3-5 μl. The inlet hole can be designed to fit a pipette tip snuglyand to create a good seal around the pipette tip, within the cone of theinlet hole. Once the pipette tip lands within the cone, the conicalshape guides the pipette and mechanically seals the combination toprovide error free dispensing or withdrawal of fluid into the cartridge.However, the cone is designed such that the sealing is reversiblebecause it is undesirable if the seal is so tight that the cartridge canbe pulled away from its tray, or location in the receiving bay, when thepipette tips are lifted after the dispensing operations.

In some embodiments, the microfluidic cartridge further comprises aregistration member 3009 that ensures that the cartridge is received bya complementary diagnostic apparatus in a single orientation, forexample, in a receiving bay of the apparatus. The registration membermay be a simple cut-out from an edge or a corner of the cartridge (asshown in FIG. 1), or may be a series of notches, wedge or curved-shapedcutouts, or some other configuration of shapes that require a uniqueorientation of placement in the apparatus.

In some embodiments, the multi-sample cartridge has a size substantiallythe same as that of a 96-well plate as is customarily used in the art.Advantageously, then, the cartridge may be used with plate handlers usedelsewhere in the art.

In some embodiments, the microfluidic cartridge comprises two or morepositioning elements, or fiducials 3010, for use when filling the valveswith thermally responsive material. The positioning elements may belocated on the substrate, typically the upper face thereof. In theembodiment of FIG. 1, they are shown on diagonally opposed corners ofthe substrate but are not limited to such positions.

After PCR has been carried out on a sample, and presence or absence of apolynucleotide of interest has been determined, it is typical that theamplified sample remains on the cartridge and that the cartridge iseither used again (if one or more lanes remain open), or disposed of.Should a user wish to run a post amplification analysis, such as gelelectrophoresis, the user may pierce a hole through the laminate of thecartridge, and recover an amount—typically about 1.5 microliter—of PCRproduct. The user may also place the individual PCR lane on a specialnarrow heated plate, maintained at a temperature to melt the wax in thevalve, and then aspirate the reacted sample from the inlet hole of thatPCR lane.

The microfluidic cartridges may also be stackable, such as for easystorage or transport, or may be configured to be received by a loadingdevice, as further described herein, that holds a plurality ofcartridges in close proximity to one another, but without being incontact with one another. In order to accomplish either or both of thesecharacteristics, the substrate may comprise two ledges or ridges, one ofeach of which is situated along each of two opposite edges of thecartridge, the ledges or ridges typically disposed on the upper side ofthe substrate. Thus, where a cartridge has a rectangular aspect(ignoring any registration member or mechanical key), the two ridges maybe situated along the long sides, or along the short sides, of thecartridge. In the embodiment of FIG. 1, the two ridges 3011 are situatedalong the long sides of the cartridge. The ridges can be, e.g., 2 mmwide, and in general are sufficiently wide to accomplish stacking. Theinlet holes are positioned sufficiently far from the ridges to preventinterference with liquid inputting operations.

In various embodiments, during transport and storage, the microfluidiccartridge can be further surrounded by a sealed pouch to reduce effectsof, e.g., water vapor. The microfluidic cartridge can be sealed in thepouch with an inert gas. The microfluidic cartridge can be disposable,such as intended for a single use.

In use, a microfluidic cartridge, as described herein, is typicallythermally associated with an array of heat sources configured to operatethe components (e.g., valves, gates, and PCR reactor) of themicrofluidic networks. In some embodiments, the heat sources arecontrolled by an operating system, which operates the device during use.The operating system includes a processor (e.g., a computer) configuredto actuate the heat sources according to a desired protocol. Processorsconfigured to operate microfluidic devices are described in, e.g., U.S.application Ser. No. 09/819,105, filed Mar. 28, 2001, which applicationis incorporated herein by reference.

The application of pressure (such as ˜1 psi) to contact the cartridge tothe heater of the instrument assists in achieving better thermal contactbetween the heater and the heat-receivable parts of the cartridge, andalso prevents the bottom laminate structure from expanding, as wouldhappen if the PCR channel was partially filled with liquid and theentrapped air would be thermally expanded during thermocycling.

Three further exemplary embodiments of a microfluidic cartridge areshown in, respectively, FIGS. 2A-2E, 3, and 4.

FIG. 2A shows a perspective view of a portion of an exemplarymicrofluidic cartridge 200 according to the present technology. Thecartridge may be referred to as a multi-lane PCR cartridge. FIGS. 2B,2C, and 2D show respectively top plan, side, and bottom plan views ofthe cartridge of FIG. 2A. Visible in FIGS. 2A-2D are variousrepresentative components of cartridge 200. In particular, theembodiment of FIGS. 2A-2D contains twelve independent sample lanes 209capable of independent (simultaneous or successive) processing ofsamples. Each lane has a dedicated pipette inlet 202. For example,sample inlet 202 is configured to accept a syringe, a pipette, or a PCRtube containing a PCR ready sample. More than one inlet 202 is shown,wherein one inlet operates in conjunction with a single lane. Variouscomponents of microfluidic circuitry in each lane are also visibleand/or shown in FIG. 2E. For example, microvalves 204 and 206 aremicrofluidic components that are parts of microfluidic circuitry in agiven lane. Also shown is an ultrafast PCR reactor 210, which, asfurther described herein, is a microfluidic channel that is long enoughto permit PCR to occur in a sample. Typical dimensions of a PCR reactorare 150μ deep by 700μ wide, and a typical volume is ˜1.6 μl. Above PCRreactor 210 is a window 212 that permits optical detection, such asdetection of fluorescence from a fluorescent substance, such as afluorogenic hybridization probe, in PCR reactor 210 when a detector issituated above window 212.

FIG. 3 shows schematically an aspect of the cartridge herein, that it isnot limited to twelve lanes. The broken section in FIG. 3 illustratesthat embodiments of the cartridge may differ in their aspect ratios soas to accommodate fewer than, or more than, twelve lanes. Numbers oflanes that may be found in embodiments of microfluidic cartridgesconsistent with the other description herein, include but are notlimited to, 4, 6, 8, 10, 15, 16, 20, 24, 30, 32, 36, 40, and 48.

FIG. 4A shows a perspective view of an exemplary microfluidic cartridge400 having 12 lanes 409, though as with other cartridges herein, othernumbers of lanes are consistent with the operation of this cartridge.The cartridge is particularly configured to accept samples from a manualpipetting device, rather than an automated pipette head. Tab 401 isoptional, and when present facilitates removal of the cartridge from areceiving bay into which it is loaded during use. Thus, a user grabs thetab 401, and pulls the cartridge out from a recessed location thathinders gripping the cartridge by is edges.

FIG. 4B shows a plan view of the underside of the embodiment of FIG. 4A,and depicts the microfluidic networks in each lane. FIG. 4C shows aperspective cutaway view of a portion of the top of the cartridge ofFIG. 4A, showing various aspects of the microfluidic circuits. FIG. 4Dshows the microfluidic circuit of a single lane of the cartridge of FIG.4A.

Microfluidic Networks

FIG. 2E (left panel) shows a plan view of a representative microfluidiccircuit found in one lane of a multi-lane cartridge such as shown inFIG. 2A. FIG. 2E (right panel) shows how the circuit is visible throughthe cartridge construction.

FIG. 4D shows a plan view of a representative microfluidic circuit foundin one lane of a multi-lane cartridge such as shown in FIGS. 4A and 4B.

Other configurations of microfluidic network, not shown in FIGS. 2E and4D, would be consistent with the function of the cartridges andapparatus described herein.

The microfluidic networks of FIGS. 2E and 4D are now described, whereinit is apparent that like reference numerals refer to like elements.Throughout the operation of cartridge 200 the fluid is manipulated as amicrodroplet (not shown in FIGS. 2E, 4D).

In sequence, sample-containing solution is introduced through liquidinlet 202 that communicates with inlet hole 203. The network optionallyincludes a bubble removal vent channel 208 into which sample flows andwhich permits adventitious air bubbles introduced into the sample duringentry to escape. Typically, when using a robotic dispenser of liquidsample (e.g., as described in U.S. patent application Ser. No.12/212,403, filed Sep. 17, 2008, and incorporated herein by reference),the volume is dispensed accurately enough that formation of bubbles isnot a significant problem, and the presence of vent channel 208 is notnecessary. Conversely, vent channel 208 finds most use when sample isintroduced into the network via manual pipetting. After passing throughthe bubble vent, if present, sample continues along a channel 216; ifthere is no bubble removal vent, as in FIG. 2E, the sample solutionflows directly from the inlet into channel 216.

Channel 216 is shown as kinked around the location of microvalve 206.Such a kink is not necessary, but merely convenient to ensure that thewhole network occupies as small an area of the microfluidic substrate asis practical.

Typically, the microfluidic network is configured so that the timerequired for a microdroplet of sample to pass from the inlet to thesecond valve is less than 50% of the time required for the sample totravel up to the exit vent. Typically, the microfluidic network isdesigned to have an increased flow resistance downstream of the twovalves without increasing the total volume of the microfluidic networkin comparison to the amount required to fill from the first valve to theend vent of the network.

Valves 204 and 206 are shown in FIG. 4D as double-valves, having asource of thermally responsive material (also referred to as atemperature responsive substance) on either side of the channel wherethey are situated. However, as shown in FIG. 2E, valves 204 and 206 mayeither or both be single valves that have a source of thermallyresponsive material on only one side of the respective channels. Valves204 and 206 are initially open, so that a microdroplet ofsample-containing fluid can be pumped into PCR reactor 210 from inlethole 202. Upon initiating of processing of the sample, the detectorpresent on top of the PCR reactor checks for the presence of liquid inthe PCR reactor, and then, if liquid is present, communicates that factto a processor controller that causes valves 204 and 206 to be closed tothereby isolate the PCR reaction mix from the channels on either side.Both valves 204 and 206 are closed prior to thermocycling to prevent anyevaporation of liquid, bubble generation, or movement of fluid from thePCR reactor, during PCR. The use of microvalves configured such asvalves 204 and 206 prevents both loss of liquid or vapor therebyenabling even a partially filled reactor to successfully complete a PCRthermocycling reaction.

The PCR reactor 210 is a microfluidic channel that is heated through aseries of cycles to carry out amplification of nucleotides in thesample, as further described herein. Typically the PCR reactor has avolume of 3-5 μl, and in particular embodiments has a volume of 4 μl, or4.5 μl. For PCR reactors of such volumes, the input volume of fluid istypically from 4 μl to 6 μl. The inside walls of the channel in the PCRreactor are made very smooth and polished to a shiny finish (forexample, using a polish such as SPI A1, SPI A2, SPI A3, SPI b1, or SPIB2) during manufacture. This is in order to minimize any microscopic airtrapping in the surface of the PCR reactor, which would causing bubblingduring the thermocycling steps. The presence of bubbles especially inthe detection region of the PCR reactor might cause a false reading for,e.g., completion of PCR. Furthermore, the PCR reactor 210 is madeshallow such that the temperature gradient across the depth of thechannel is minimized. The region 212 of the substrate above PCR reactor210 permits a detector to monitor progress of the reaction and also todetect fluorescence from a probe that binds to a quantity of amplifiednucleotide. The region 212 is made of thinner material than the rest ofthe cartridge so as to permit the PCR reactor to be more responsive to aheating cycle (for example, to rapidly heat and cool betweentemperatures appropriate for denaturing and annealing steps), and so asto reduce glare, autofluorescence, and undue absorption of fluorescence.

In some embodiments, an end vent 214 prevents a user from introducingany excess amount of liquid into the microfluidic cartridge, such as caneasily be done during manual pipetting. As further described herein, anend vent is typically covered by a membrane. In other embodiments, anoverflow chamber 229 plays a role of containing any sample from spillingover to unintended parts of the cartridge. An overflow chamber istypically configured to capture 1-2 microliters of fluid. In still otherembodiments, both an end vent and an overflow chamber 229 are present,and such that usually the end vent follows downstream of the overflowchamber. A user may input sample volumes as small as an amount to fillfrom the bubble removal vent (where present) to the middle of the PCRreactor, or up to valve 204 or beyond valve 204. The end vent, overflowchamber, or combination thereof, serve to prevent excess fluid fromflooding the microfluidic networks.

Also shown in FIG. 2E are residual portions 220 and 222, and 224 ofstructures that are removed from the cartridge substrate aftermanufacture. The presence of the structures during manufacture is tohelp with proper filling of the cartridge when molten plastic is flowedinto the mold (mold flow). Items 220 and 224 are often called “dogbones”because of their shape. Still other shapes are of course possible andconsistent with methods of manufacture and use, as described herein.

Also shown in FIG. 4D is a projection of a spacer element 219. This itemis also shown in FIG. 4C. It supports a label affixed over the vent sucha way that it does not touch the vent.

In various embodiments, the microfluidic network can optionally includeat least one reservoir configured to contain waste.

Table 1 outlines volumes, pumping pressures, and operation timesassociated with various components of a microfluidic network, as foundin microfluidic cartridges described herein.

TABLE 1 Pumping Displacement Operation Pressure Volume Time of OperationMixing ~2 psi 10-25 μl 1-2 minutes displacements Moving valve ~1-2 psi<1 μl 5-15 seconds wax plugs Operation Pump Used Pump Design PumpActuation Mixing Expancel Pump Same as above Same as above displacementsMoving valve Thermopneumatic 1 μl of Heat trapped air wax plugs pumptrapped air to ~70-90 C.Valves

A valve is a microfluidic component that has a normally open stateallowing material to pass along a channel from a position on one side ofthe valve (e.g., upstream of the valve) to a position on the other sideof the valve (e.g., downstream of the valve). Various valves findapplication in the microfluidic networks herein.

An exemplary double valve 3100 is shown in FIGS. 5A and 5B. A doublevalve has two channels 3101, 3103, one on either side of the channel3102 whose flow it regulates, whereas a single valve, such as in FIGS.6A, 6B, has just one channel, disposed on one side of the channel whoseflow it regulates. Channels 3101, 3103 are referred to as loadingchannels because a thermally responsive substance (TRS) is loaded intosuch channels, for example via fluidly connected valve inlets 3105 and3106.

Upon actuation, e.g., by application of heat, the valve transitions to aclosed state that prevents material, such as a microdroplet of PCR-readysample, from passing along the channel 3102 from one side of the valveto the other. For example, a valve includes one or more masses of athermally responsive substance (TRS) that is relatively immobile at afirst temperature and more mobile at a second temperature. Actuation ofthe valve involves transitioning the TRS from a temperature at or lowerthan the first temperature to a temperature that is at or higher thanthe second temperature.

A mass of TRS can be an essentially solid mass or an agglomeration ofsmaller particles that cooperate to obstruct the passage upon actuation.Examples of TRS's include a eutectic alloy (e.g., a solder), wax (e.g.,an olefin), polymers, plastics, and combinations thereof. The first andsecond temperatures are insufficiently high to damage materials, such aspolymer layers of a microfluidic cartridge in which the valve issituated. Generally, the second temperature is less than about 90° C.and the first temperature is less than the second temperature (e.g.,about 70° C. or less).

For each mass of TRS associated with a valve, a chamber is in gaseouscommunication with the mass. Upon heating gas (e.g., air) in thechamber(s) and heating the one or more masses of TRS to the secondtemperature, gas pressure within a chamber moves the corresponding massinto the channel obstructing material from passing therealong. Othervalves of the microfluidic network have the same structure and operatein the same fashion as the valves described herein.

In order to make the valve sealing very robust and reliable, the flowchannel 3102 at the valve junction is made narrow (150 μm wide and 150μm deep or narrower) and the constricted portion of the channel is madeat least 0.5 or 1 mm long such that the wax seals up a long narrowchannel thereby reducing any leakage of fluid sample through the wallsof the channel. In the case of a bad seal, there is leakage of fluidaround the walls of the channel, past the wax. So the flow channel isnarrowed as much as possible, and made longer, e.g., as long as ˜1 mm.

The valve is operated by heating trapped pockets of air in the valveinlets (also referred to as wax-loading ports), which forces the TRSforwards in a manner so that it does not come back to its originalposition. In this way, both air and TRS are heated during operation ofthe valve.

FIGS. 6A and 6B show embodiments of single valves whose structures areconsistent with use in the microfluidic networks described herein.

In various embodiments, the microfluidic network can include a singlevalve, also referred to as a bent valve, as shown in FIG. 6A (as asingle valve) to reduce the footprint of the valve on the cartridge andhence reduce cost per part for manufacturing highly dense microfluidicsubstrates. In the valve of FIG. 6A, the loading hole for TRS is in thecenter of the valve; the structures at either end are an inlet and anoutlet and are shown for illustrative purposes only.

In various embodiments, the network can include a curved valve as shownin FIG. 6B, also as a single valve, in order to reduce the effectivecross-section of the microvalve, also enabling manufacture of cheaperdense microfluidic devices.

Vents

A hydrophobic vent (e.g., vent 3200 in FIG. 7) is a structure thatpermits gas to exit a channel while limiting (e.g., preventing) liquidfrom exiting the channel. Typically, hydrophobic vents include a layerof porous hydrophobic material (e.g., a porous filter such as a poroushydrophobic membrane from Osmonics) that defines a wall of the channel.As discussed herein, hydrophobic vents can be used to position amicrodroplet of sample at a desired location within a microfluidicnetwork. In embodiments such as shown in FIGS. 4A-4D, the membrane ispresent as a layer (such as of a oleophobic or hydrophobic material)positioned underneath one or more of the labels, and configured to coverthe vent channels of microfluidic substrate. It can be applied usingheat bonding. A suitable material is a 0.2 to 1.0 micron pore-sizemembrane of modified polytetrafluorethylene (typically between about 25and about 100 microns thick).

The hydrophobic vents of the cartridge are preferably constructed sothat the amount of air that escapes through them is maximized whileminimizing the volume of the channel below the vent surface.Accordingly, it is preferable that the vent is constructed so as to havea hydrophobic membrane of large surface area and a shallow cross sectionof the microchannel below the vent surface. An exemplary membrane is aPTFE membrane having 0.22μ pore size. Other pore sizes are consistentwith operation of the vents herein, and according to application.

Bubble removal hydrophobic vents typically have a length of at leastabout 2.5 mm (e.g., at least about 5 mm, at least about 7.5 mm) along achannel. The length of the hydrophobic vent is typically at least about5 times (e.g., at least about 10 times, at least about 20 times) largerthan a depth of the channel 3201 within the hydrophobic vent. Forexample, in some embodiments, the channel depth within the hydrophobicvent is about 300 microns or less (e.g., about 250 microns or less,about 200 microns or less, about 150 microns or less). Bubble vents areoptional in the microfluidic networks of the microfluidic cartridgesdescribed herein.

The depth of the channel within the hydrophobic vent is typically about75% or less (e.g., about 65% or less, about 60% or less) of than thedepth of the channel upstream and downstream of the hydrophobic vent.For example, in some embodiments the channel depth within thehydrophobic vent is about 150 microns and the channel depth upstream anddownstream of the hydrophobic vent is about 250 microns.

A width of the channel within the hydrophobic vent is typically at leastabout 25% wider (e.g., at least about 50% wider) than a width of thechannel upstream from the vent and downstream from the vent. Forexample, in an exemplary embodiment, the width of the channel within thehydrophobic vent is about 400 microns and the width of the channelupstream and downstream from the vent is about 250 microns.

The vent of FIG. 7 is a hydrophobic vent having a linear channel 3201,connected via an inlet 3203 and an outlet 3204 to the rest of themicrofluidic network. The right hand panel of FIG. 7 shows the vent intop plan view, and indicates where the membrane is situated.

Manufacturing Process for Cartridge

FIG. 8 shows a flow-chart 2800 depicting an assembly process for anexemplary cartridge as further described herein. It would be understoodby one of skill in the art, both that various steps may be performed ina different order from that set forth in FIG. 8, and additionally thatany given step may be carried out by alternative methods to those setforth in the figure. It would also be understood that, where separatesteps are illustrated for carrying out two or more functions, suchfunctions may be performed synchronously and combined into single stepsand still be consistent with the overall process described herein.

At 2802, a laminate layer is applied to a microfluidic substrate thathas previously been engineered to have a plurality of microfluidicnetworks constructed in it; edges are trimmed from the laminate wherethey spill over the bounds of the substrate. The laminate seals invarious components of the microfluidic networks.

At 2804, a thermally responsive substance such as wax is dispensed andloaded into the microvalves of the microfluidic network in themicrofluidic substrate from the opposite side of the substrate to thatsealed by the laminate. An exemplary process for carrying this out isfurther described herein.

At 2806, the cartridge is inspected to ensure that wax from 2804 isloaded properly and that the laminate from 2802 adheres properly to themicrofluidic substrate. If a substrate does not satisfy either or bothof these tests, it is discarded. If substrates repeatedly fail either orboth of these tests, then the wax dispensing, or laminate applicationsteps, as applicable, are reviewed.

Optionally, at 2808, for cartridge embodiments that employ a hydrophobicvent, a hydrophobic vent membrane is applied to, and heat bonded to, thetop of the microfluidic substrate over the wax-loaded valves, and on theopposite face of the substrate from the laminate. Edges of the membranethat are in excess of the boundary of the substrate are trimmed.

Optionally, at 2810 if a vent membrane has been applied (2808), theassembly is inspected to ensure that the hydrophobic vent membrane isbonded well to the microfluidic substrate without heat-clogging themicrofluidic channels. If any of the channels is blocked, or if the bondbetween the membrane and the substrate is imperfect, the assembly isdiscarded, and, in the case of repeated discard events, the foregoingprocess is reviewed.

At 2812, a label layer is applied to the top of the microfluidicsubstrate, situated to cover the valves. It would be understood that amultiple label parts, instead of a single layer, may be devised tofulfill both of these roles. In the embodiments in which a membrane hasbeen affixed, the label(s) may comprise a portion that protects the ventmembrane. The label typically has identifying indicia, such as abarcode, printed on it before it is applied to the top of the substrate.

At 2814 optionally, additional indicia are printed or applied to thelabel layer to show identifying characteristics, such as a barcode #,lot # and expiry date on the cartridge. Typically one or more of thelabels has a space and a writable surface that permits a user to make anidentifying annotation on the label, by hand.

At 2816, to facilitate transport and delivery to a customer, assembledand labeled cartridges are stacked and pack cartridges in groups, suchas groups of 24 or 25, or groups of 10, or groups of 20, or groups of50. Preferably the packaging is via an inert and/or moisture-freemedium.

Wax Loading in Valves

FIGS. 9A and 9B show how a combination of controlled hot drop dispensingvia a heated microchannel device of the right dimensions and geometry isused to accurately load wax into a microchannel of a microfluidiccartridge to form a valve. The heated dispenser head (not shown in FIGS.10A, 10B) can be accurately positioned over an inlet hole of themicrochannel in the microfluidic device, and can dispense molten waxdrops in volumes as small as 75 nanoliters with an accuracy of +/−20%.The inlet hole of the microchannel device is dimensioned in such a waythat the droplet of 75 nl can be accurately shot to the bottom of theinlet hole using, for example, compressed air, or in a manner similar toan inkjet printing method, or by using a DJ-9000 manufactured byAsymtek, as further described herein.

The microchannel device is maintained at a temperature above the meltingpoint of the wax thereby permitting the wax to stay in a molten stateimmediately after it is dispensed. After the drop falls to the bottom ofthe inlet hole, the molten wax is drawn into the narrow channel bycapillary action. The volume of the narrow section is designed to beapproximately equal to a maximum typical amount that is dispensed intothe inlet hole.

The valve, shown in cross-section in FIGS. 9A and 9B, contains a stepbetween the inlet and the loading channel. The step facilitatesretention of the thermally responsive substance.

Exemplary Wax-Deposition Process

Deposition of wax in valves of the microfluidic network, as at step 2804(FIG. 8) may be carried out with the exemplary equipment shown in FIGS.10A and 10B. The DispenseJet Series DJ-9000, FIGS. 10A and 10B (explodedview) is a non-contact dispenser that provides high-speed delivery andexceptional volumetric control for various fluids, including surfacemount adhesive, underfill, encapsulants, conformal coating, UVadhesives, and silver epoxy.

The DJ-9000 jets in tight spaces as small as 200 micrometers and createsfillet wet-out widths as small as 300 micrometers on the dispensed sideof a substrate such as a die. It dispenses fluid either as discrete dotsor a rapid succession of dots to form a 100-micron (4 mil) diameterstream of fluid from the nozzle. It is fully compatible with othercommercially available systems such as the Asymtek Century C-718/C-720,Millennium M-2000, and Axiom X-1000 Series Dispensing Systems.

A DJ-9000 is manufactured by Asymtek under manufacturing quality controlstandards aim to provide precise and reliable performance.Representative specifications of the apparatus are as follows.

Characteristic Specification Size Width: 35 mm Height: 110 mm Depth: 100mm Weight 400 grams - dry Feed Tube Assembly Nylon - FittingPolyurethane - Tube Fluid Chamber Type 303 Stainless Steel Seat andNozzle 300/400 Series S/S, Carbide Needle Assembly 52100 Bearing Steel -Shaft Hard Chrome Plate Carbide - Tip Fluid Seal PEEK/Stainless SteelFluid Chamber 0-Ring Ethylene Propylene Jet Body 6061-T6 Aluminum NickelPlated Needle Assembly Bearings PEEK Thermal Control Body 6061-T6Aluminum Nickel Plated Reservoir Holder Acetyl Reservoir Size 5, 10, or30 cc (0.17, 0.34, or 1.0 oz) Feed Tube Assembly Fitting Female Luer perANSI/HIMA MD70.1-1983 Maximum Cycle Frequency 200 Hz. Minimum Valve AirPressure 5.5 bar (80 psi) Operating Noise Level 70 db* Solenoid 24 VDC,12.7 Watts Thermal Control Heater 24 VDC, 14.7 Watts, 40 ohms ThermalControl RTD 100 ohm, platinum Maximum Heater Set Point 80 C. *At MaximumCycle Rate

The DJ-9000 has a normally closed, air-actuated, spring-returnmechanism, which uses momentum transfer principles to expel precisevolumes of material. Pressurized air is regulated by a high-speedsolenoid to retract a needle assembly 2903 from seat 2901. Fluid, fedinto the fluid chamber 2902 from the fluid reservoir 2904, flows overthe seat. When the air is exhausted, the needle travels rapidly to theclosed position, displacing fluid through the seat and nozzle in theform of a droplet. Multiple droplets fired in succession can be used toform larger dispense volumes and lines when combined with the motion ofa dispenser robot that controls motion and positioning of the dispensehead.

The equipment has various adjustable features: The following featuresaffect performance of the DJ-9000 and are typically adjusted to fitspecific process conditions.

Fluid Pressure should be set so that fluid fills to the seat, but shouldnot be influential in pushing the fluid through the seat and nozzle. Ingeneral, higher fluid pressure results in a larger volume of materialjetted.

The stroke adjustment knob 2905 controls the travel distance of theneedle assembly 2903. For example, the control can be turnedcounterclockwise to increase needle assembly travel, or turned clockwiseto decrease travel. An increase of travel distance will often result ina larger volume of material jetted.

The solenoid valve 2906 controls the valve operation. When energized, itallows air in the jet air chamber to compress a spring 2907 and therebyraise the needle assembly. When de-energized, the air is released andthe spring forces the piston down so that the needle tip contacts theseat.

The seat and nozzle geometry are typically the main factors controllingdispensed material volume. The seat and nozzle size are determined basedon the application and fluid properties. Other parameters are adjustedin accordance with seat and nozzle choices. Available seat and nozzlesizes are listed in the table hereinbelow.

Fluid temperature often influences fluid viscosity and flowcharacteristics. The DJ-9000 is equipped with a thermal control assembly2908 that assures a constant fluid temperature.

In addition to the DJ-9000 hardware configuration and settings, Dot andLine Parameters can be set in a software program (for example, onereferred to as FmNT) to control the size and quality of dots and linesdispensed.

Packaging

The microfluidic cartridge described herein may be provided in aconvenient package containing multiple cartridges, and made easilyaccessible, but suitably protected during carriage. In someimplementations, the packaging comprises a plastic pouch to protect thecartridges from moisture, but this is not a requirement. The packagingmaterial in general comprise one or more of paper, foam, and cardboard.In some implementations, the packaging facilitates lifting a stack, suchas 24 cartridges, for easy transfer into a cartridge auto-loader, asdescribed elsewhere herein.

Apparatus Overview

The microfluidic cartridges as described herein find application toanalyzing any nucleic acid containing sample for any purpose, including,but not limited to, genetic testing, and clinical testing for variousinfectious diseases in humans, and food testing, for example ofagricultural products.

The microfluidic cartridges herein are configured for use with adiagnostic apparatus. Such an apparatus is described in U.S. patentapplication Ser. No. 12/173,023, filed Jul. 14, 2008, incorporatedherein by reference. Salient features of such an apparatus are nowdescribed herein. However, it would be understood that cartridges suchas those herein could be configured for use with other types ofapparatus not otherwise described herein, for example an apparatus thatis configured to just perform PCR on samples that had previously beenbrought into PCR-ready form by some other apparatus, or manually.

The apparatus of FIG. 11 is configured to act on a disposablemicrofluidic cartridge containing multiple sample lanes in parallel, andcomprises a reusable instrument platform that can actuate on-cartridgeoperations, can detect and analyze the products of the PCR amplificationin each of the lanes separately, in all simultaneously, or in groupssimultaneously, and, optionally, can display the results on a graphicaluser interface.

A schematic overview of an apparatus 981 for carrying out automatedsample preparation and diagnostic testing on multiple samples inparallel, according to steps exemplified elsewhere herein, is shown inFIG. 11. The geometric arrangement of the components of system 981 isexemplary and not intended to be limiting. In overview, multiple samplesare introduced into the apparatus and individually placed into a formsuitable for applying PCR to amplify one or nucleotides containedtherein. The PCR-ready samples are transferred to a microfluidiccartridge that is acted on by the apparatus and caused to amplifynucleotides in the samples by carrying out PCR thereon, whilst in thecartridge. Results of the amplifications can be detected while theamplified nucleotides are in the cartridge.

A processor 980, such as a microprocessor, is configured to controlfunctions of various components of the system as shown, and is therebyin communication with each such component requiring control, for examplevia a bus. It is to be understood that many such control functions canoptionally be carried out manually, and not under control of theprocessor. Furthermore, the order in which the various functions aredescribed, in the following, is not limiting upon the order in which theprocessor executes instructions when the apparatus is operating. Asuitable processor 980 can be designed and manufactured according to,respectively, design principles and semiconductor processing methodsknown in the art.

Processor 980 can be configured to accept user instructions from aninput device 984, where such instructions may include instructions tostart analyzing the sample, and choices of operating conditions.Processor 980 can be also configured to communicate with a display 982,so that, for example, information about an analysis is transmitted tothe display and thereby communicated to a user of the system. Suchinformation includes but is not limited to one or more of: the currentstatus of the apparatus; progress of PCR thermocycling; and a warningmessage in case of malfunction of either system or cartridge.Additionally, processor 980 may transmit one or more questions to bedisplayed on display 982 that prompt a user to provide input in responsethereto. Thus, in certain embodiments, input 984 and display 982 areintegrated with one another.

Processor 980 can be optionally further configured to transmit resultsof an analysis to an output device 986 such as a printer, a visualdisplay such as display 982 or a second display, a display that utilizesa holographic projection, or a speaker, or a combination thereof.Processor 980 can be still further optionally connected via acommunication interface such as a network interface to a computernetwork 988.

Processor 980 can be further configured to control various aspects ofsample preparation and diagnosis, as follows in overview. In FIG. 1, theapparatus 981 is configured to operate in conjunction with acomplementary rack 970. Apparatus 981 may be capable of receivingmultiple racks, such as 1, 2, 3, 4, or 6 racks.

Embodiments of rack 970 are further described in U.S. patent applicationSer. Nos. 12/173,023, filed by Express Mail on Jul. 14, 2008 (andentitled “Integrated Apparatus for Performing Nucleic Acid Extractionand Diagnostic Testing on Multiple Biological Samples”, in the name ofWilliams, et al.), and 12/178,584, filed on Jul. 23, 2008, and entitled“Rack For Sample Tubes And Reagent Holders”, in the name of Duffy, etal., both of which are incorporated herein by reference in theirentireties. A rack 970 is itself configured to receive a number ofbiological samples 996, such as nucleic-acid containing samples, in aform suitable for work-up and subsequent diagnostic analysis, and anumber of holders 972—as further described herein, such as in connectionwith FIG. 2, that are equipped with various reagents, pipette tips andreceptacles. The rack is configured so that, during sample work-up,samples are processed in the respective holders, the processingincluding being subjected, individually, to heating and cooling viaheater assembly 977.

The heating functions of the heater assembly 977 can be controlled bythe processor 980. Heater assembly 977 operates in conjunction with aseparator 978, such as a magnetic separator, that also can be controlledby processor 980 to move into and out of close proximity to one or moreprocessing chambers associated with the holders 972, wherein particlessuch as magnetic particles are present. Assembly 977 and separator 978are further described in U.S. patent application Ser. No. 12/178,586,filed on Jul. 23, 2008, and entitled “Integrated Heater and MagneticSeparator”, in the name of Handique, which is incorporated herein byreference in its entirety.

Processor 980 can be configured to receive data about a sample to beanalyzed, e.g., from a sample reader 990, which may be a barcode reader,an optical character reader, or an RFID scanner (radio frequency tagreader). Thus, sample reader 990 is configured to transmit identifyingindicia about the sample, and in some instances the holder, to processor980. In some embodiments, the sample reader is movable from one sampleposition to another. In some embodiments a sample reader is attached tothe liquid dispenser 976 and can thereby read indicia about a sampleabove which the liquid dispenser is situated. In other embodiments thesample reader is not attached to the liquid dispenser and isindependently movable, under control of the processor.

Liquid dispenser 976, which similarly can be controlled by processor 980and is further described herein, is configured to carry out various suckand dispense operations on respective samples in rack 970, and fluidsand reagents in the holders 972, to achieve extraction of nucleic acidfrom the samples. Liquid dispenser 976 can carry out such operations onmultiple holders simultaneously, and is further described herein.

In the embodiment of a diagnostic apparatus shown in FIG. 1, a cartridge994 is received in bay 992. The receiving bay is in communication with aheater 998 that itself can be controlled by processor 980 in such a waythat specific regions of the cartridge 994 are heated at specific timesduring analysis. Liquid dispenser 976 is configured to take aliquots offluid containing nucleic acid extracted from one or more samples anddirect them to one or more respective inlets in cartridge 994. Cartridge994 is configured to amplify, such as by providing chambers for carryingout PCR on, the respective nucleic acids. The processor is alsoconfigured to control and receive data from a detector 999 that receivesan indication of a diagnosis from the cartridge 994. The diagnosis canbe transmitted to the output device 986 and/or the display 982, asdescribed hereinabove.

Embodiments of the apparatus shown in outline in FIG. 1, as with otherexemplary embodiments described herein, are advantageous because they donot require locations within the apparatus suitably configured forstorage of reagents. Therefore, the apparatus in FIG. 1 isself-contained and operates in conjunction with holders 972 andcartridges 994, wherein the holders are pre-packaged with reagents, suchas in locations within it dedicated to reagent storage, and wherein thecartridges are supplied with separately packaged PCR reagentsappropriate for mixing with PCR-ready sample, prior to introduction intothe cartridge.

The apparatus of FIG. 1 may be configured to carry out operation in asingle location, such as a laboratory setting, or may be portable sothat they can accompany, e.g., a physician, or other healthcareprofessional, who may visit patients at different locations. Theapparatus is typically provided with a power-cord so that it can acceptAC power from a mains supply or generator. The apparatus may also beconfigured to operate by using one or more batteries and therefore isalso typically equipped with a battery recharging system, and variouswarning devices that alert a user if battery power is becoming too lowto reliably initiate or complete a diagnostic analysis.

The apparatus of FIG. 1 may further be configured, in other embodiments,for multiplexed sample analysis and/or analysis of multiple batches ofsamples, where, e.g., a single rack holds a single batch of samples.Each component shown in FIG. 1 may therefore be independently present asmany times as there are batches of samples (or some fraction thereof),though the multiple instances of the various components may beconfigured in a common housing.

In various embodiments, preparation of a PCR-ready sample for use insubsequent diagnosis using the apparatus as further described herein caninclude one or more of the following steps: contacting a neutralizedpolynucleotide sample with a PCR reagent mixture comprising a polymeraseenzyme and a plurality of nucleotides (in some embodiments, the PCRreagent mixture can further include a positive control plasmid and afluorogenic hybridization probe selective for at least a portion of theplasmid); in some embodiments, the PCR reagent mixture can be in theform of one or more lyophilized pellets, as stored in a receptacle on aholder, and the method can further include reconstituting the PCR pelletwith liquid to create a PCR reagent mixture solution.

The apparatus herein can be configured to run on a laboratory benchtop,or similar environment, and can test approximately 45 samples per hourwhen run continuously throughout a normal working day. Results fromindividual raw samples are typically available in less than 1 hour.

FIGS. 12A and 12B show views of an exemplary diagnostic apparatus 3000incorporating various elements of FIG. 11. Shown in FIG. 12A, a frontplan view of apparatus 3000 has a hinged cover 3010, shown in a closedposition, bearing an optional clear window 3012 (that provides a userwith an at-a-glance indication of the apparatus' status) and a handle3014 that facilitates opening and closing of the cover.

Shown in FIG. 12B is a front plan view of apparatus 3000 with cover 3010moved to an open position revealing certain elements of the interior3020 of the apparatus. Aspects of the interior of the apparatus that arevisible in the view of FIG. 12B include: two removable racks 970, eachbearing 12 holders 972, a liquid dispenser 976, mounted on a gantry2102, and a receiving bay 992 for holding a microfluidic cartridge, asfurther described herein.

Various aspects of an apparatus configured for use with a microfluidiccartridge are now further described.

Cartridge Receiving Bay

In some embodiments, an apparatus includes a bay configured toselectively receive a microfluidic cartridge, at least one heat sourcethermally coupled to the bay and coupled to a processor as furtherdescribed herein, such that the heat source is configured to heatindividual sample lanes in the cartridge, and the processor isconfigured to control application of heat to the individual samplelanes, separately, in all simultaneously, or in groups simultaneously.

In various embodiments, the microfluidic networks in the cartridge canbe configured to couple heat from an external heat source, such as inthe receiving bay, to a sample mixture comprising PCR reagent andPCR-ready, such as neutralized, polynucleotide sample and caused toundergo thermal cycling conditions suitable for creating PCR ampliconsfrom the PCR-ready sample.

FIG. 13 shows an interior view of an exemplary apparatus, showing a rackholding a number of sample tubes and reagent holders, and a cartridge994 situated in receiving bay 992.

FIG. 14 shows a perspective view of an exemplary cartridge 200 thatcontains multiple sample lanes, and a removeable receptacle shown astray 110 that, optionally, can accommodate cartridge 200 prior toinsertion of the cartridge in a receiving bay in an apparatus. Tray 110makes it easier for the user to place the cartridge into a receiving baysuch as in a diagnostic apparatus. Alignment of the cartridge into aremoveable receptacle such as tray 110 is often easier than trying toseat the cartridge directly into a recessed area. Thus, tray 110typically has a registration member that engages with a registrationmember, such as a notch or corner-cut-out, on the cartridge, and tray110 also has raised portions that can be held in a user's hand duringplacement and removal of the tray+cartridge into a receiving bay. Tray110 is also typically configured so that it holds the cartridge butstructural elements of the tray do not interpose themselves between thecartridge and, e.g., a detector or a heating element, when the cartridgeis in use.

The bay can be a portion of the apparatus that is configured toselectively receive the microfluidic cartridge. For example, the bay andthe microfluidic cartridge can be complementary in shape so that themicrofluidic cartridge is selectively received in, e.g., a singleorientation. For example, the microfluidic cartridge can have aregistration member that fits into a complementary feature of the bay.The registration member can be, for example, a cut-out on an edge of thecartridge, such as a corner that is cut-off, or one or more notches thatare made on one or more of the sides. By selectively receiving thecartridge, the bay can help a user to place the cartridge so that theapparatus can properly operate on the cartridge. In this way, error-freealignment of cartridges can be achieved. Moreover, the cartridge can bedesigned to be slightly smaller than the receiving bay by approximately200-300 micron for easy placement and removal of the cartridge. Theapparatus can further include a sensor configured to sense whether themicrofluidic cartridge is selectively received

The bay can also be configured so that various components of theapparatus that can operate on the microfluidic cartridge (heat sources,detectors, force members, and the like) are positioned to properlyoperate on the microfluidic cartridge while the cartridge is received inthe bay. For example, a contact heat source can be positioned in the baysuch that it can be thermally coupled to a distinct location at amicrofluidic cartridge that is selectively received in the receivingbay.

Alternatively, in connection with alignment of microheaters in theheater module with corresponding heat-requiring microcomponents (such asvalves, pumps, gates, reaction chambers, etc), the microheaters can bedesigned to be slightly bigger than the heat requiring microfluidiccomponents so that even though the cartridge may be off-centered fromthe heater, the individual microfluidic components can still functioneffectively.

The heat source can be, for example, a heat source such as a resistiveheater or network of resistive heaters, a reversible heat source such asa liquid-filled heat transfer circuit or a thermoelectric element, aradiative heat source such as a xenon lamp, and the like.

In preferred embodiments, the at least one heat source can be a contactheat source selected from a resistive heater (or network thereof), aradiator, a fluidic heat exchanger and a Peltier device. The contactheat source can be configured at the receiving bay to be thermallycoupled to one or more distinct locations of a microfluidic cartridgereceived in the bay, whereby the distinct locations are selectivelyheated. At least one additional contact heat source can be included,wherein the contact heat sources are each configured at the bay to beindependently thermally coupled to a different distinct location in amicrofluidic cartridge received in the bay, whereby the distinctlocations are independently heated. The contact heat source can beconfigured to be in direct physical contact with a distinct location ofa microfluidic cartridge received in the bay. In various embodiments,each contact source heater can be configured to heat a distinct locationhaving an average diameter in 2 dimensions from about 1 millimeter (mm)to about 15 mm (typically about 1 mm to about 10 mm), or a distinctlocation having a surface area of between about 1 mm² about 225 mm²(typically between about 1 mm² and about 100 mm², or in some embodimentsbetween about 5 mm² and about 50 mm²).

In various embodiments, at least one heat source can be a radiative heatsource configured to direct heat to a distinct location of amicrofluidic cartridge received in the receiving bay. The bottom surfaceof the cartridge typically mates with the heating surface to form a snugfit.

In various embodiments, the apparatus includes one or more force membersthat are configured to apply force to thermally couple the at least oneheat source to at least a portion of the microfluidic cartridge receivedin the bay. The one or more force members can be configured to operate amechanical member at the microfluidic cartridge. Typically themechanical member at the cartridge is held in place by a motor thatapplies pressure to the member. In some embodiments, a force member canbe manually operated. At least one force member can be mechanicallycoupled to a lid at the receiving bay, whereby operation of the lidoperates the force member. In various embodiments, the force applied bythe motor or the one or more force members can result in an averagepressure at an interface between a portion of the receiving bay and aportion of the microfluidic cartridge of about 1 psi. The application offorce is important to ensure consistent thermal contact between theheater wafer and the PCR reactor and microvalves in the microfluidiccartridge.

In various embodiments, the apparatus can further include a lid at thereceiving bay, the lid being operable to at least partially excludeambient light from the bay. The lid can be, for example, a sliding lidbut is more typically a hinged, sprung, or motor operated lid that comesdown on the cartridge from above. The lid can include the opticaldetector. A major face of the lid at the bay can vary from planarity byless than about 100 micrometers, for example, less than about 25micrometers. The lid can be configured to be removable from theapparatus. The lid can include a latching member that ensures that thelid is securely closed before amplification reactions are applied to thesamples in the cartridge.

FIG. 15 shows a schematic cross-sectional view of a part of an apparatusas described herein, showing input of sample into an inlet 202 ofcartridge 200 via a pipette tip 10 (such as a disposable pipette)attached to an automated dispensing head. Although not shown, there areas many inlets 202 as samples to be input into cartridge 200. Inlet 202is preferably configured to receive a pipette or the bottom end of a PCRtube and thereby accept sample for analysis with minimum waste, and withminimum introduction of air. Cartridge 200 is disposed on top of and incontact with a heater substrate 400. Read head 300 is positioned abovecartridge 200 and a cover for optics 310 restricts the amount of ambientlight that can be detected by the read head.

The detector can be, for example, an optical detector, as furtherdescribed herein. For example, the detector can include a light sourcethat selectively emits light in an absorption band of a fluorescent dye,and a light detector that selectively detects light in an emission bandof the fluorescent dye, wherein the fluorescent dye corresponds to afluorescent polynucleotide probe or a fragment thereof. Alternatively,for example, the optical detector can include a bandpass-filtered diodethat selectively emits light in the absorption band of the fluorescentdye and a bandpass filtered photodiode that selectively detects light inthe emission band of the fluorescent dye; or for example, the opticaldetector can be configured to independently detect a plurality offluorescent dyes having different fluorescent emission spectra, whereineach fluorescent dye corresponds to a fluorescent polynucleotide probeor a fragment thereof; or for example, the optical detector can beconfigured to independently detect a plurality of fluorescent dyes at aplurality of different locations on a microfluidic cartridge, whereineach fluorescent dye corresponds to a fluorescent polynucleotide probeor a fragment thereof in a different sample.

Advantageously, in some embodiments the receiving bay design allows easyplacement of the microfluidic cartridge, such as by a user, or anauto-loading device as further described herein. Such a design alsoaccommodates multiple sample pipetting of liquid using a roboticdispenser, and optical detection in situ. Furthermore, it is typicallyeasier to move a cartridge and heater in and out of position than adetector.

Heater Configurations to Ensure Uniform Heating of a Region

Another aspect of the apparatus described herein relates to uniformcontrol of the heating of a region of a microfluidic network, the regionincluding to one or more microfluidic components. In an exemplaryembodiment, multiple heaters can be configured to simultaneously anduniformly heat a region, such as the PCR reaction zone, of amicrofluidic network in the microfluidic cartridge.

In preferred embodiments, a microfluidic cartridge having one or moremicrofluidic networks, each comprising one or more microfluidiccomponents, is brought into contact with a heat source, within asuitably configured apparatus. The heat source is configured so thatparticular heating elements are situated to heat specific components ofthe microfluidic networks of the cartridge.

FIG. 16 shows a cross-sectional view of an exemplary microfluidiccartridge, situated in a receiving bay, to show relative location of PCRchamber 901 in relation to the heaters when the cartridge is placed inthe instrument. PCR chamber 901 is shown in a substrate layer 907 of thecartridge. A laminate layer 905 of the cartridge is directly under thePCR chamber 901.

Two long heaters 909 and 911 that run alongside (when viewed from above)PCR chamber 901 are situated in a substrate layer 913 of the receivingbay, directly under and in contact with the laminate layer of thecartridge. The heaters are photolithographically defined and etchedmetal layers of gold (typically about 3,000 Å thick). Layers of 400 Å ofTiW are deposited on top and bottom of the gold layer to serve as anadhesion layer. The substrate used can be a glass, fused silica, orquartz wafer having a thickness of 0.2-1 mm, such as 0.4 mm, 0.5 mm or0.7 mm, or 1 mm. A thin electrically-insulative layer of 2 μm siliconoxide serves as an insulative layer on top of the metal layer.Additional thin electrically insulative layers such as 2-4 μm ofparylene may also be deposited on top of the silicon oxide surface.

Referring to FIGS. 17A and 17B, the PCR reaction chamber 1001 isconfigured with a long side and a short side, each with an associatedheating element. The apparatus therefore preferably includes fourheaters disposed along the sides of, and configured to heat, the PCRreaction zone: long top heater 1005, long bottom heater 1003, short leftheater 1007, and short right heater 1009. The small gap between long topheater 1005 and long bottom heater 1003 results in a negligibletemperature gradient (less than 1° C. across the width of the PCRchannel at any point along the length of the PCR reaction zone) andtherefore an effectively uniform temperature throughout the PCR reactionzone. The heaters on the short edges of the PCR reactor provide heat tocounteract the gradient created by the two long heaters from the centerof the reactor to the edge of the reactor.

It would be understood by one of ordinary skill in the art that stillother configurations of one or more heater(s) situated about a PCRreaction zone are consistent with the methods and apparatus describedherein. For example, a ‘long’ side of the reaction zone can beconfigured to be heated by two or more heaters. Specific orientationsand configurations of heaters are used to create uniform zones ofheating even on substrates having poor thermal conductivity because thepoor thermal conductivity of glass, or quartz, or fused silicasubstrates is utilized to help in the independent operation of variousmicrofluidic components such as valves and independent operation of thevarious PCR lanes.

In preferred embodiments, each heater has an associated temperaturesensor. In the embodiment of FIG. 17A, a single temperature sensor 1011is used for both long heaters. A temperature sensor 1013 for short leftheater, and a temperature sensor 1015 for short right heater are alsoshown. The temperature sensor in the middle of the reactor is used toprovide feedback and control the amount of power supplied to the twolong heaters, whereas each of the short heaters has a dedicatedtemperature sensor placed adjacent to it in order to control it. Asfurther described herein, temperature sensors are preferably configuredto transmit information about temperature in their vicinity to theprocessor at such times as the heaters are not receiving current thatcauses them to heat. This can be achieved with appropriate control ofcurrent cycles.

In order to reduce the number of sensor or heater elements required tocontrol a PCR heater, the heaters may be used to sense as well as heat,and thereby obviate the need to have a separate dedicated sensor foreach heater. In another embodiment, each of the four heaters may bedesigned to have an appropriate wattage, and connect the four heaters inseries or in parallel to reduce the number ofelectronically-controllable elements from 4 to just 1, thereby reducingthe burden on the electronics.

FIG. 17B shows expanded views of heaters and temperature sensors used inconjunction with a PCR reaction zone of FIG. 17A. Temperature sensors1001 and 1013 are designed to have a room temperature resistance ofapproximately 200-300 ohms. This value of resistance is determined bycontrolling the thickness of the metal layer deposited (e.g., a sandwichof 400 Å TiW/3000 Å Au/400 Å TiW), and etching the winding metal line tohave a width of approximately 10-25 μm and 20-40 mm length. The use ofmetal in this layer gives it a temperature coefficient of resistivity ofthe order of 0.5-20° C./ohms, preferably in the range of 1.5-3° C./ohms.Measuring the resistance at higher temperatures will enabledetermination of the exact temperature of the location of these sensors.

The configuration for uniform heating, shown in FIG. 17A for a singlePCR reaction chamber, can be applied to a multi-lane PCR cartridge inwhich multiple independent PCR reactions occur.

Each heater can be independently controlled by a processor and/orcontrol circuitry used in conjunction with the apparatus describedherein. FIG. 18 shows thermal images, from the top surface of amicrofluidic cartridge having heaters configured as in FIGS. 17A and17B, when each heater in turn is activated, as follows: (A): Long Toponly; (B) Long Bottom only; (C) Short Left only; (D) Short Right only;and (E) All Four Heaters on. Panel (F) shows a view of the reaction zoneand heaters on the same scale as the other image panels in FIG. 18. Alsoshown in the figure is a temperature bar.

Use of Cutaways in Cartridge and Heater Substrates to Improve Rate ofCooling During PCR Cycling

During a PCR amplification of a nucleotide sample, a number of thermalcycles are carried out. For improved efficiency, the cooling betweeneach application of heat is preferably as rapid as possible. Improvedrate of cooling can be achieved with various modifications to thecartridge and to the heating substrate, as shown in FIGS. 19A-19C.

One way to achieve rapid cooling is to cutaway portions of themicrofluidic cartridge substrate, as shown in FIG. 19A. The upper panelof FIG. 19A is a cross-section of an exemplary microfluidic cartridgeand heater unit taken along the dashed line A-A′ as marked on the lowerpanel of FIG. 19A. PCR reaction chamber 901, and representative heaters1003 are shown. Also shown are two cutaway portions, one of whichlabeled 1201, that are situated alongside the heaters that are situatedalong the long side of the PCR reaction zone. Cutaway portions such as1201 reduce the thermal mass of the cartridge, and also permit air tocirculate within the cutaway portions. Both of these aspects permit heatto be conducted away quickly from the immediate vicinity of the PCRreaction zone. A further cutout portion 919 in the cartridge directlyabove the PCR chamber serves to both reduce thermal mass and backgroundfluorescence. The latter is useful because, since cutout 919 is situateddirectly above the PCR chamber, the amount of material between thechamber and a fluorescence detector is reduced. Other configurations ofcutouts, such as in shape, position, and number, are consistent with thepresent technology.

Another way to achieve rapid cooling is to cutaway portions of theheater substrate, as shown in FIG. 19B. The lower panel of FIG. 19B is across-section of an exemplary microfluidic cartridge and heatersubstrate 921 taken along the dashed line A-A′ as marked on the upperpanel of FIG. 19B. PCR reaction chamber 901 situated in substrate 923 ofthe cartridge, and representative heaters 1003 are shown. Laminate layer921 of the cartridge is situated on top of heater substrate 921. Thecartridge has a cutout 919 above the PCR chamber, as described inconnection with FIG. 19A. Also shown in FIG. 19B are four cutawayportions, one of which labeled 1205, that are situated alongside theheaters that are situated along the long side of the PCR reaction zone.(Cutaways 1205 are shown cross-hatched in the upper panel of FIG. 19B,even though they are shown as clear (no cross-hatching) in the lowerpanel.) Cutaway portions such as 1205 reduce the thermal mass of theheater substrate, and also permit air to circulate within the cutawayportions. Both of these aspects permit heat to be conducted away quicklyfrom the immediate vicinity of the PCR reaction zone. Four separatecutaway portions are shown in FIG. 19B. Multiple cutaway portions aretypically used so that control circuitry to the various heaters is notdisrupted. Other configurations of cutouts, such as in shape, position,and number, are consistent with the present technology. These cutoutsmay be created by a method selected from: selective etching using wetetching processes, deep reactive ion etching, selective etching usingCO₂ laser or femtosecond laser (to prevent surface cracks or stress nearthe surface), selective mechanical drilling, selective ultrasonicdrilling, or selective abrasive particle blasting. Care has to be takento maintain mechanical integrity of the heater while reducing as muchmaterial as possible.

FIG. 19C shows a combination of cutouts in cartridge (comprisingsubstrate 923 and laminate layer 925) and heater substrate 921, and useof ambient air cooling to increase the cooling rate during the coolingstages of thermocycling. A substantial amount of cooling happens byconvective loss from the bottom surface of the heater surface to ambientair. The driving force for this convective loss is the differential intemperatures between the heater substrate surface (typically made ofglass) and the air temperature. By decreasing the ambient airtemperature by use of, for example, a peltier cooler 1207, the rate ofcooling can be increased. The convective heat loss may also be increasedby keeping the air at a velocity higher than zero. Peltier cooler 1207is situated beneath a printed circuit board, layer 927 as shown incross-section in FIG. 19C. Peltier cooler comprises a cooling surface1209, a number of p-n junctions 1211 on a substrate 1213. The substrateitself can be mounted on a finned heat sink 1215, in which are one ormore cutouts 1217 to facilitate cooling.

An example of thermal cycling performance obtained with a configurationas described herein, is shown in FIG. 20 for a protocol that is set toheat up to 92° C., and stay there for 1 second, then cool to 62° C., andstay for 10 seconds. Cycle time is about 29 seconds, with 8 secondsrequired to heat from 62° C. and stabilize at 92° C., and 10 secondsrequired to cool from 92° C., and stabilize at 62° C.

Highly Multiplexed Embodiments

Embodiments of the apparatus and cartridge described herein may beconstructed that utilize high-density microfluidic circuitry on a singlecartridge that thereby permit processing of multiple samples inparallel, or in sequence, on a single cartridge. Preferred numbers ofsuch multiple samples include 36, 40, 48, 50, 64, 72, 80, 84, 96, and100, but it would be understood that still other numbers are consistentwith the apparatus and cartridge herein, where deemed convenient andpractical.

Accordingly, different configurations of lanes, sample inlets, andassociated heater networks that can facilitate processing such numbersof samples on a single cartridge are within the scope of the instantdisclosure.

In an exemplary embodiment, shown in FIGS. 21A-21C, a highly multiplexedcartridge has 48 lanes. FIG. 21A shows a plan view of a representative48-lane cartridge. FIG. 21B shows, in close up, an exemplary spacing ofvalves and lanes in adjacent lanes of a multi-sample microfluidiccartridge.

FIG. 21C shows an inlet configuration for accepting 48 samples. Theinlet configuration is compatible with an automatic pipetting machinethat has dispensing heads situated at a 8 mm spacing. For example, sucha machine having 4 heads can load 4 inlets of the cartridge of FIGS. 21Aand 21C at once, in 12 discrete steps.

Each lane of the cartridge has a microfluidic network that includes aPCR chamber and has independently controllable valves in one or morechannels, with 2 banks of thermocycling protocols per channel, as shownin FIG. 22A. In the embodiment in FIG. 22A, the heaters are arranged inthree arrays. Heaters in two separate glass regions only apply heat tovalves in the microfluidic networks in each lane. Because of the lowthermal conductivity of glass, the individual valves may be heatedseparately from one another. This permits samples to be loaded into thecartridge at different times, and passed to the PCR reaction chambersindependently of one another. The PCR heaters are mounted on a siliconsubstrate—and are not readily heated individually, but thereby permitbatch processing of PCR samples, where multiple samples from differentlanes are amplified by the same set of heating/cooling cycles. In someembodiments, the PCR heaters are arranged in 2 banks (the heater arrayson the left and right are not in electrical communication with oneanother), thereby permitting a separate degree of sample control.

FIGS. 22A and 22B show heater arrays of the exemplary cartridge of FIG.21A in, respectively, plan view, and in close-up.

FIGS. 23A-23C show various views of an embodiment of aradially-configured highly-multiplexed cartridge, having a number ofinlets, microfluidic lanes, and PCR reaction zones.

The various embodiments shown in FIGS. 21A-23C are compatible withliquid dispensers, receiving bays, and detectors that are configureddifferently from the specific examples described herein.

In another embodiment (not shown in the FIGs.), a cartridge andapparatus is configured so that the read-head does not cover the sampleinlets, thereby permitting loading of separate samples while othersamples are undergoing PCR thermocycling.

Heater Multiplexing (Under Software Control)

Another aspect of the apparatus described herein, relates to a methodfor controlling the heat within the system and its components. Themethod leads to a greater energy efficiency of the apparatus describedherein, because not all heaters are heating at the same time, and agiven heater is receiving current for only part of the time.

Generally, the heating of microfluidic components, such as a PCRreaction zone, is controlled by passing currents through suitablyconfigured microfabricated heaters, as illustrated in FIG. 4E. Theheating can be further controlled by periodically turning the current onand off with varying pulse width modulation (PWM), wherein pulse widthmodulation refers to the on-time/off-time ratio for the current. Thecurrent can be supplied by connecting a microfabricated heater to a highvoltage source (for example, 30V), which can be gated by the PWM signal.In some embodiments, the device includes 48 PWM signal generators.Operation of a PWM generator includes generating a signal with a chosen,programmable period (the end count) and granularity. For instance, thesignal can be 4000 μs (micro-seconds) with a granularity of 1 μs, inwhich case the PWM generator can maintain a counter beginning at zeroand advancing in increments of 1 μs until it reaches 4000 μs, when itreturns to zero. Thus, the amount of heat produced can be adjusted byadjusting the end count. A high end count corresponds to a greaterlength of time during which the microfabricated heater receives currentand therefore a greater amount of heat produced.

In various embodiments, the operation of a PWM generator can alsoinclude a programmable start count in addition to the aforementioned endcount and granularity. In such embodiments, multiple PWM generators canproduce signals that can be selectively non-overlapping (e.g., bymultiplexing the on-time of the various heaters) such that the currentcapacity of the high voltage power is not exceeded. Multiple heaters canbe controlled by different PWM signal generators with varying start andend counts. The heaters can be divided into banks, whereby a bankdefines a group of heaters of the same start count.

For example, 36 PWM generators can be grouped into six different banks,each corresponding to a certain portion of the PWM cycle (500 ms forthis example). The end count for each PWM generator can be selectivelyprogrammed such that not more than six heaters will be on at any giventime. A portion of a PWM cycle can be selected as dead time (count 3000to 4000 for this example) during which no heating takes place andsensitive temperature sensing circuits can use this time to sense thetemperature. The table below represents a PWM cycle for the foregoingexample:

Start Count End Count Max End Count Bank 1 PWM generator#1 0 150 500 PWMgenerator#2 0 220 500 . . . . . . . . . PWM generator#6 0 376 500 Bank 2PWM generator#7 500 704 1000 PWM generator#8 500 676 1000 . . . . . . .. . . . . PWM generator#12 500 780 1000 Bank 3 PWM generator#13 10001240 1500 PWM generator#14 1000 1101 1500 . . . . . . . . . . . . PWMgenerator#18 1000 1409 1500 Bank 4 PWM generator#19 1500 1679 2000 PWMgenerator#20 1500 1989 2000 . . . . . . . . . . . . PWM generator#241500 1502 2000 Bank 5 PWM generator#25 2000 2090 2500 PWM generator#262000 2499 2500 . . . . . . . . . . . . PWM generator#30 2000 2301 2500Bank 6 PWM generator#31 2500 2569 3000 PWM generator#32 2500 2790 3000 .. . . . . . . . . . . PWM generator#36 2500 2678 3000Use of Detection System to Measure/Detect Fluid in PCR Chamber

The apparatus optionally has a very sensitive fluorescence detector thatis able to collect fluorescence light from the PCR chamber 210 of amicrofluidic cartridge. Further aspects of such a detector are describedin U.S. patent application Ser. No. 12/173,023, filed Jul. 14, 2008, andincorporated herein by reference. Such a detector is used to monitor theprogress of PCR, and can also be used to detect the presence of liquidin the chamber, a measurement that determines whether or not to carryout a PCR cycle on that particular chamber. One way to accomplish thisdetermination is to take a background reading prior to filling thechamber with liquid. Another reading is taken after operations have beenperformed that should result in filling the PCR chamber with liquid. Thepresence of liquid alters the fluorescence reading from the chamberrelative to an empty chamber. A programmable threshold value is used totune an algorithm programmed into the processor (for example, the secondreading has to exceed the first reading by 20%). If the two readings donot differ beyond the programmed margin, the liquid is deemed to nothave entered the chamber, and a PCR cycle is not initiated for thatchamber. Instead, a warning is issued to a user.

Liquid Dispenser

The microfluidic cartridge is configured to receive sample(s) via theone or more inlets, and delivered by a liquid dispenser. A suitableliquid dispenser for use with the apparatus herein is described in U.S.patent application Ser. No. 12/212,403, filed Sep. 17, 2008, andincorporated herein by reference.

In various embodiments, preparation of a PCR-ready sample for use insubsequent diagnosis using the apparatus as further described herein,can include one or more of the following steps: contacting a neutralizedpolynucleotide sample with a PCR reagent mixture comprising a polymeraseenzyme and a plurality of nucleotides (in some embodiments, the PCRreagent mixture can further include a positive control plasmid and afluorogenic hybridization probe selective for at least a portion of theplasmid); in some embodiments, the PCR reagent mixture can be in theform of one or more lyophilized pellets, as stored in a receptacle on aholder, and the method can further include reconstituting the PCR pelletwith liquid to create a PCR reagent mixture solution. Various, such asone or more, of the liquid transfer operations associated with theforegoing steps can be accomplished by an automated pipette head.

During sample preparation, the liquid dispenser is configured to carryout fluid transfer operations on two or more holders simultaneously, andcan be mounted on a gantry having three degrees of translationalfreedom.

The liquid dispenser typically comprises a number of individually sprungheads, wherein each head is configured to accept a pipette tip from theone or more pipette tips in a holder, and to control dispense operationswith that pipette tip. For example, a typical liquid dispenser comprisesfour individually sprung heads, but it is to be understood that thedispenser is not limited to this number. For example, other numbersinclude 2, 3, 5, 6, 8, 10, or 12. The liquid dispenser can be furtherconfigured such that no two heads accept pipette tips from the sameholder. The individually sprung heads may be arranged in parallel to oneanother, but may be configured in other arrangements.

The liquid dispenser is further configured to aspirate or dispense fluidin connection with analysis or preparation of solutions of two or moresamples. The liquid dispenser is also configured to dispense liquid intoa microfluidic cartridge. Additionally, the liquid dispenser isconfigured to accept or dispense, in a single operation, an amount of1.0 ml of fluid or less, such as an amount of fluid in the range 10 nl-1ml.

Typically when transferring a sample containing extracted nucleic acidfrom a pipette tip to an inlet on the microfluidic cartridge, say usinga liquid dispenser, a volume of air is simultaneously introduced intothe microfluidic network that is connected to the inlet, the volume ofair being typically between about 0.5 mL and about 5 mL, but dependingon the volume of the pipette tip. Presence of air in the microfluidicnetwork, however, is not essential to operation of the cartridgedescribed herein.

Proper alignment of pipette tips attached to the dispense head with theinlets on the cartridge can be achieved with a motorized alignmentplate, as further described in U.S. patent application Ser. No.12/212,403, filed Sep. 17, 2008, and incorporated herein by reference.

Cartridge Autoloader

In certain embodiments of the apparatus described elsewhere herein,microfluidic cartridges are stored in a loading device and automaticallydelivered to a receiving bay when needed. Such an arrangement, which maybe referred to as an amplification-detection system, permits multiplesamples to be analyzed, in batches, and with minimal interruptionbetween successive cartridges.

An exemplary embodiment of an amplification-detection system 2900 foruse with a microfluidic cartridge is shown in FIG. 24. The system 2900performs and automates the process of PCR on multiple nucleic-acidcontaining samples, on a cartridge, in parallel. The system 2900comprises a repository 2907 for unused microfluidic cartridges, acartridge autoloader, a receiving bay 2902 for a microfluidic cartridge,a detector 2908, and a waste tray 2903 configured to receive usedmicrofluidic cartridges. In one embodiment, the cartridge autoloadercomprises a cartridge pack 2901, and a cartridge pusher 2904.

The system 2900, for illustration purposes, is configured so that amicrofluidic cartridge moves in a plane and linearly from the repository2907 to the receiving bay 2902, to the waste bin 2903, but it need notbe so arranged. For example, the waste cartridge bin 2903 can be alignedorthogonally, or any angle thereof, to the receiving bay, such asdisposed behind it. Alternatively, each element (cartridge autoloader2901, receiving bay 2902, and waste cartridge bin 2903) can beconfigured in a step-wise manner where the cartridge pack 2901 is on thesame, higher or lower level than the amplification-detection system 2902and the amplification-detection system 2902 is on the same, higher orlower level than the waste cartridge bin 2903. Another configurationcould be that each of the three elements is not arranged linearly but atan angle to one another, although within the same plane.

FIG. 24 illustrates the cartridge pack 2901 and the waste cartridge bin2903 below the plane of the receiving bay, and a detection system 2908above the plane. This configuration is exemplary and it would beunderstood that some of these elements may be positioned alternatelyabove or below the plane in other embodiments.

FIG. 25 illustrates a repository for unused microfluidic cartridges. Therepository can be configured to accept a number of individually stackedand individually loaded cartridges, or can be configured to accept apack of cartridges 2901, where it is to be understood that a pack is aset of two or more cartridges that are stored and transported togetherwith one another, even though each will be used separately. An exemplarycartridge pack has 24 cartridges. The repository may consist of a cage2910 of any material that may or may not be transparent. For example itmay be made of metal or plastic. The cartridge pack 2901 is not limitedto twenty-four cartridges 2906 per pack but may contain any number from2 to 100. For example, other numbers such as 2, 4, 8, 10, 12, 16, 20,30, 36, 40, 48, 50, or 64 are possible numbers of cartridges per pack.Similarly, the repository may be configured to accept those numbers ofcartridges, when individually stacked, rather than being in a pack.

In some embodiments, as in FIG. 25, each cartridge 2906, individuallystacked, rests so that ridges along two sides of the cartridge aresupported on ledges 2911 that protrude from the cage 2910. However,other configurations are possible. For example, a cartridge 2906 mayrest on recessed grooves made within the interior surfaces of cage 2910.Furthermore, the cartridge pack 2901 may not need to be placed in a cage2910. The cartridge pack 2901 may itself include the necessaryconnections to bind securely to the apparatus and to load the cartridges2906.

FIG. 26 is an illustration of an exemplary initial loading position of acartridge pack 2901 in a depository when samples are loaded in thetopmost cartridge in the pack. FIG. 26 shows the cartridge pack 2901below a plane that contains a cartridge pusher. In other embodiments,the cartridge pack 2901 may be above the plane of a cartridge pusherwhere the pusher pushes the lowest cartridge out from the holder; orpartly above and partly below in a holder 2920 where a cartridge pusherpushes a cartridge from the middle of the cartridge pack 2901. In theembodiment shown in FIG. 26, a topmost cartridge 106 is pushed along twoguide rails 2905. Alternatively, there may be more or fewer guide rails(such as one or three) or no guide rails at all so long as a cartridge2906 can be caused to move to other required positions with reliabilityand accuracy of positioning.

An exemplary cartridge pusher 2904 is shown in FIG. 27. The cartridgepusher 2904 pushes a cartridge 2906 along guide rails 2905, which allowsa cartridge 2906 to travel to pre-calibrated positions by the mechanismof a stepper motor 2930. However, it would be understood that themechanism of transporting the cartridge 2906 is not limited to a steppermotor 2930 and thus other mechanisms are also consistent with thecartridge pusher 2904 as described herein.

FIG. 28 shows a used cartridge 2906 that has been pushed by thecartridge pusher 2904 into the waste cartridge bin 2903 after a PCRprocess has been completed, such as on multiple samples in thecartridge. The embodiment shows a lipped handle 2940 that facilitateseasy handling, such as emptying, of the bin 2903. However, it would beunderstood that the handle 2904 is not limited to the style and shapeshown.

An exemplary cartridge pack 2901, before and after multiple PCRprocesses are completed, is shown in FIG. 29. After the cartridge pusher2904 pushes a cartridge 2906 out of the cartridge pack 2901, a spring2950 at the bottom of the cartridge pack pushes against the lowersurface of the stack of cartridges and causes the topmost cartridge tobe made available for sample injection. The spring 2950 is not limitedin number or type. Thus although a single helical or coiled spring isshown, it is consistent with the description herein that more than onehelical or coiled springs could be used, such as 2, 3, or 4, and thatalternatively a sprung metal strip, or several strips, could be used.Alternatively another mechanism for forcing the cartridges upwards couldbe deployed, such as a pneumatic, hydraulic, or inflatable pressurizedcontainer, could be utilized.

The cartridge pushing mechanism can also be made to not only push thecartridge from the autoloader box to the detection position, but also beused to move it back to the autoloading position. This will enableunused lanes in the microfluidic cartridge to be used in the next PCRrun.

The cartridge autoloading box is also designed so that once all thecartridges are used, the box can be easily recycled or new cartridgesadded to it. This reduces the cost to the customer and the manufacturer.

It is to be noted that microfluidic cartridges, as further describedherein, that have a raised lip along their edges to permit ease ofstacking and/or storage in a pack or an auto-loader are particularlyadvantageous because the raised lips also introduce a stiffness into thecartridges and assist in keeping the fluid inlets on one cartridge awayfrom those on another cartridge during storage and transport. The raisedregions, which need not only be lips along each edge of a cartridge,also help minimize friction between the lower surface of one cartridgeand the upper surface of another during transport and use.

Sample Preparation, and Reagents

In various embodiments, the PCR-ready sample injected into the cartridgecan include a PCR reagent mixture comprising a polymerase enzyme, and anucleotide or a plurality of nucleotides from a biological sample inquestion. The PCR reagent mixture can be in the form of one or morelyophilized pellets prior to mixing with the sample, and the steps bywhich the PCR-ready sample is prepared can involve contacting the PCRpellet with liquid to create a PCR reagent mixture solution. In yetanother embodiment, each of the PCR chambers in the cartridge may havedried down or lyophilized ASR reagents preloaded such that the user onlyneeds to input prepared polynucleotide sample into the cartridge. Inanother embodiment, the PCR chambers of the cartridge may have only theapplication-specific probes and primers premeasured and preloaded, andthe user inputs a sample mixed with the PCR reagents.

In various embodiments, the PCR-ready sample further includes a samplebuffer, and at least one probe that is selective for a polynucleotidesequence, e.g., the polynucleotide sequence that is characteristic of apathogen selected from the group consisting of gram positive bacteria,gram negative bacteria, yeast, fungi, protozoa, and viruses.

In various embodiments, the sample can include at least one probe thatcan be selective for a polynucleotide sequence. The probe can be afluorogenic hybridization probe. The fluorogenic hybridization probe caninclude a polynucleotide sequence coupled to a fluorescent reporter dyeand a fluorescence quencher dye.

In various embodiments, the PCR ready sample can further include apositive control plasmid and a fluorogenic hybridization probe selectivefor at least a portion of the plasmid, and the microfluidic cartridgecan be configured to allow independent optical detection of thefluorogenic hybridization probe and the plasmid fluorogenichybridization probe.

In various embodiments, the microfluidic cartridge can accommodate anegative control polynucleotide, wherein the microfluidic network can beconfigured to independently carry out PCR on each of a neutralizedpolynucleotide sample and a negative control polynucleotide with the PCRreagent mixture under thermal cycling conditions suitable forindependently creating PCR amplicons of the neutralized polynucleotidesample and PCR amplicons of the negative control polynucleotide.

Each lane of a multi-lane cartridge as described herein can perform tworeactions because of the presence of two fluorescence detection systemsper lane. A variety of combinations of reactions can be performed in thecartridge, such as two sample reactions in one lane, a positive controland a negative control in two lanes, such as adjacent lanes; or a samplereaction and an internal control in one lane and a negative control in aseparate lane.

Carrying out PCR on a PCR-ready sample can include heating the PCRreagent mixture and the neutralized polynucleotide sample under thermalcycling conditions suitable for creating PCR amplicons from theneutralized polynucleotide sample; contacting the neutralizedpolynucleotide sample or a PCR amplicon thereof with at least one probethat is selective for a polynucleotide sequence; independentlycontacting each of the neutralized polynucleotide sample and a negativecontrol polynucleotide with the PCR reagent mixture under thermalcycling conditions suitable for independently creating PCR amplicons ofthe neutralized polynucleotide sample and PCR amplicons of the negativecontrol polynucleotide; and/or contacting the neutralized polynucleotidesample or a PCR amplicon thereof and the negative control polynucleotideor a PCR amplicon thereof with at least one probe that is selective fora polynucleotide sequence.

In various embodiments, a method of using the apparatus described hereincan further include one or more of the following steps: determining thepresence of a polynucleotide sequence in the biological sample, thepolynucleotide sequence corresponding to the probe, if the probe isdetected in the neutralized polynucleotide sample or a PCR ampliconthereof; determining a contaminated result if the probe is detected inthe negative control polynucleotide or a PCR amplicon thereof; and/or insome embodiments, wherein the PCR reagent mixture further comprises apositive control plasmid and a plasmid probe selective for at least aportion of the plasmid, the method further including determining that aPCR amplification has occurred if the plasmid probe is detected.

In various embodiments, the probes used can be selective for apolynucleotide sequence that is characteristic of an organism, forexample any organism that employs deoxyribonucleic acid or ribonucleicacid polynucleotides. Thus, the probe can be selective for any organism.Suitable organisms include mammals (including humans), birds, reptiles,amphibians, fish, domesticated animals, wild animals, extinct organisms,bacteria, fungi, viruses, plants, and the like. The probe can also beselective for components of organisms that employ their ownpolynucleotides, for example mitochondria. In some embodiments, theprobe is selective for microorganisms, for example, organisms used infood production (for example, yeasts employed in fermented products,molds or bacteria employed in cheeses, and the like) or pathogens (e.g.,of humans, domesticated or wild mammals, domesticated or wild birds, andthe like). In some embodiments, the probe is selective for organismsselected from the group consisting of gram positive bacteria, gramnegative bacteria, yeast, fungi, protozoa, and viruses.

In various embodiments, the probe can be selective for a polynucleotidesequence that is characteristic of an organism selected from the groupconsisting of Staphylococcus spp., e.g., S. epidermidis, S. aureus,Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin-resistantStaphylococcus; Streptococcus(e.g., α, β or γ-hemolytic, Group A, B, C,D or G) such as S. pyogenes, S. agalactiae; E. faecalis, E. durans, andE. faecium (formerly S. faecalis, S. durans, S. faecium);nonenterococcal group D streptococci, e.g., S. bovis and S. equines;Streptococci viridans, e.g., S. mutans, S. sanguis, S. salivarius, S.mitior, A. milleri, S. constellatus, S. intermedius, and S. anginosus;S. iniae; S. pneumoniae; Neisseria, e.g., N. meningitides, N.gonorrhoeae, saprophytic Neisseria sp; Erysipelothrix, e.g., E.rhusiopathiae; Listeria spp., e.g., L. monocytogenes, rarely L. ivanoviiand L. seeligeri; Bacillus, e.g., B. anthracis, B. cereus, B. subtilis,B. subtilus niger, B. thuringiensis; Nocardia asteroids; Legionella,e.g., L. pneumonophilia, Pneumocystis, e.g., P. carinii;Enterobacteriaceae such as Salmonella, Shigella, Escherichia (e.g., E.coli, E. coli O157:H7); Klebsiella, Enterobacter, Serratia, Proteus,Morganella, Providencia, Yersinia, and the like, e.g., Salmonella, e.g.,S. typhi S. paratyphi A, B (S. schottmuelleri), and C (S. hirschfeldii),S. dublin S. choleraesuis, S. enteritidis, S. typhimurium, S.heidelberg, S. newport, S. infantis, S. agona, S. montevideo, and S.saint-paul; Shigella e.g., subgroups: A, B, C, and D, such as S.flexneri, S. sonnei, S. boydii, S. dysenteriae; Proteus (P. mirabilis,P. vulgaris, and P. myxofaciens), Morganella (M. morganii); Providencia(P. rettgeri, P. alcalifaciens, and P. stuartii); Yersinia, e.g., Y.pestis, Y. enterocolitica; Haemophilus, e.g., H. influenzae, H.parainfluenzae H. aphrophilus, H. ducreyi; Brucella, e.g., B. abortus,B. melitensis, B. suis, B. canis; Francisella, e.g., F. tularensis;Pseudomonas, e.g., P. aeruginosa, P. paucimobilis, P. putida, P.fluorescens, P. acidovorans, Burkholderia (Pseudomonas) pseudomallei,Burkholderia mallei, Burkholderia cepacia and Stenotrophomonasmaltophilia; Campylobacter, e.g., C. fetus fetus, C. jejuni, C. pylori(Helicobacter pylori); Vibrio, e.g., V. cholerae, V. parahaemolyticus,V. mimicus, V. alginolyticus, V. hollisae, V. vulnificus, and thenonagglutinable vibrios; Clostridia, e.g., C. perfringens, C. tetani, C.difficile, C. botulinum; Actinomyces, e.g., A. israelii; Bacteroides,e.g., B. fragilis, B. thetaiotaomicron, B. distasonis, B. vulgatus, B.ovatus, B. caccae, and B. merdae; Prevotella, e.g., P. melaninogenica;genus Fusobacterium; Treponema, e.g. T. pallidum subspecies endemicum,T. pallidum subspecies pertenue, T. carateum, and T. pallidum subspeciespallidum; genus Borrelia, e.g., B burgdorferi; genus Leptospira;Streptobacillus, e.g., S. moniliformis; Spirillum, e.g., S. minus;Mycobacterium, e.g., M. tuberculosis, M. bovis, M. africanum, M. aviumM. intracellulare, M. kansasii, M. xenopi, M. marinum, M. ulcerans, theM. fortuitum complex (M. fortuitum and M. chelonei), M. leprae, M.asiaticum, M. chelonei subsp. abscessus, M. fallax, M. fortuitum, M.malmoense, M. shimoidei, M. simiae, M. szulgai, M. xenopi; Mycoplasma,e.g., M. hominis, M. orale, M. salivarium, M. fermentans, M. pneumoniae,M. bovis, M. tuberculosis, M. avium, M. leprae; Mycoplasma, e.g., M.genitalium; Ureaplasma, e.g., U. urealyticum; Trichomonas, e.g., T.vaginalis; Cryptococcus, e.g., C. neoformans; Histoplasma, e.g., H.capsulatum; Candida, e.g., C. albicans; Aspergillus sp; Coccidioides,e.g., C. immitis; Blastomyces, e.g. B. dermatitidis; Paracoccidioides,e.g., P. brasiliensis; Penicillium, e.g., P. mameffei; Sporothrix, e.g.,S. schenckii; Rhizopus, Rhizomucor, Absidia, and Basidiobolus; diseasescaused by Bipolaris, Cladophialophora, Cladosporium, Drechslera,Exophiala, Fonsecaea, Phialophora, Xylohypha, Ochroconis,Rhinocladiella, Scolecobasidium, and Wangiella; Trichosporon, e.g., T.beigelii; Blastoschizomyces, e.g., B. capitatus; Plasmodium, e.g., P.falciparum, P. vivax, P. ovale, and P. malariae; Babesia sp; protozoa ofthe genus Trypanosoma, e.g., T. cruzi; Leishmania, e.g., L. donovani, L.major L. tropica, L. mexicana, L. braziliensis, L. viannia braziliensis;Toxoplasma, e.g., T. gondii; Amoebas of the genera Naegleria orAcanthamoeba; Entamoeba histolytica; Giardia lamblia; genusCryptosporidium, e.g., C. parvum; Isospora belli; Cyclosporacayetanensis; Ascaris lumbricoides; Trichuris trichiura; Ancylostomaduodenale or Necator americanus; Strongyloides stercoralis Toxocara,e.g., T. canis, T. cati; Baylisascaris, e.g., B. procyonis; Trichinella,e.g., T. spiralis; Dracunculus, e.g., D. medinensis; genus Filarioidea;Wuchereria bancrofti; Brugia, e.g., B. malayi, or B. timori; Onchocercavolvulus; Loa loa; Dirofilaria immitis; genus Schistosoma, e.g., S.japonicum, S. mansoni, S. mekongi, S. intercalatum, S. haematobium;Paragonimus, e.g., P. Westermani, P. Skriabini; Clonorchis sinensis;Fasciola hepatica; Opisthorchis sp; Fasciolopsis buski; Diphyllobothriumlatum; Taenia, e.g., T. saginata, T. solium; Echinococcus, e.g., E.granulosus, E. multilocularis; Picornaviruses, rhinoviruses echoviruses,coxsackieviruses, influenza virus; paramyxoviruses, e.g., types 1, 2, 3,and 4; adnoviruses; Herpesviruses, e.g., HSV-1 and HSV-2;varicella-zoster virus; human T-lymphotrophic virus (type I and typeII); Arboviruses and Arenaviruses; Togaviridae, Flaviviridae,Bunyaviridae, Reoviridae; Flavivirus; Hantavirus; Viral encephalitis(alphaviruses [e.g., Venezuelan equine encephalitis, eastern equineencephalitis, western equine encephalitis]); Viral hemorrhagic fevers(filoviruses such as Ebola, Marburg and arenaviruses such as Lassa,Machupo); Smallpox (variola); retroviruses e.g., human immunodeficiencyviruses 1 and 2; human papillomavirus (HPV) types 6, 11, 16, 18, 31, 33,and 35.

In various embodiments, the probe can be selective for a polynucleotidesequence that is characteristic of an organism selected from the groupconsisting of Pseudomonas aeruginosa, Proteus mirabilis, Klebsiellaoxytoca, Klebsiella pneumoniae, Escherichia coli, AcinetobacterBaumannii, Serratia marcescens, Enterobacter aerogenes, Enterococcusfaecium, vancomycin-resistant enterococcus (VRE), Staphylococcus aureus,methecillin-resistant Staphylococcus aureus (MRSA), Streptococcusviridans, Listeria monocytogenes, Enterococcus spp., Streptococcus GroupB, Streptococcus Group C, Streptococcus Group G, Streptococcus Group F,Enterococcus faecalis, Streptococcus pneumoniae, Staphylococcusepidermidis, Gardenerella vaginalis, Micrococcus sps., Haemophilusinfluenzae, Neisseria gonorrhoeee, Moraxella catarrahlis, Salmonellasps., Chlamydia trachomatis, Peptostreptococcus productus,Peptostreptococcus anaerobius, Lactobacillus ferrmentum, Eubacteriumlentum, Candida glabrata, Candida albicans, Chlamydia spp., Camplobacterspp., Salmonella spp., smallpox (variola major), Yersina Pestis, HerpesSimplex Virus I (HSV I), and Herpes Simplex Virus II (HSV II).

In various embodiments, the probe can be selective for a polynucleotidesequence that is characteristic of Group B Streptococcus.

Computer Program Product

In various embodiments, a computer program product, such as a computerreadable medium, for use with the apparatus herein includes computerreadable instructions thereon for execution by a processor in connectionwith operating the apparatus.

In various embodiments, the computer program product can include one ormore instructions to cause the system to: output an indicator of theplacement of the microfluidic cartridge in the bay; read a sample labelor a microfluidic cartridge label; output directions for a user to inputa sample identifier; output directions for a user to load a sampletransfer member with the PCR-ready sample; output directions for a userto introduce the PCR-ready sample into the microfluidic cartridge;output directions for a user to place the microfluidic cartridge in thereceiving bay; output directions for a user to close the lid to operatethe force member; output directions for a user to pressurize thePCR-ready sample in the microfluidic cartridge by injecting thePCR-ready sample with a volume of air between about 0.5 mL and about 5mL; and output status information for sample progress from one or morelanes of the cartridge.

In various embodiments, the computer program product can include one ormore instructions to cause the system to: heat the PCR ready-sampleunder thermal cycling conditions suitable for creating PCR ampliconsfrom the neutralized polynucleotide; contact the neutralizedpolynucleotide sample or a PCR amplicon thereof with at least one probethat is selective for a polynucleotide sequence; independently contacteach of the neutralized polynucleotide sample and a negative controlpolynucleotide with the PCR reagent mixture under thermal cyclingconditions suitable for independently creating PCR amplicons of theneutralized polynucleotide sample and PCR amplicons of the negativecontrol polynucleotide; contact the neutralized polynucleotide sample ora PCR amplicon thereof and the negative control polynucleotide or a PCRamplicon thereof with at least one probe that is selective for apolynucleotide sequence; output a determination of the presence of apolynucleotide sequence in the biological sample, the polynucleotidesequence corresponding to the probe, if the probe is detected in theneutralized polynucleotide sample or a PCR amplicon thereof; and/oroutput a determination of a contaminated result if the probe is detectedin the negative control polynucleotide or a PCR amplicon thereof.

In various embodiments, the computer readable instructions areconfigured to independently actuate one or more microfluidic components(such as valves) of each lane in the microfluidic cartridge,independently of one another, and for causing a detector to measurefluorescence from the PCR reaction zones.

In various embodiments, the computer readable instructions present auser with the option to either get results from all samples as quicklyas possible, or from a first batch of samples as quickly as possible andthe subsequent batch later on.

EXAMPLES Example 1 Exemplary 3-Layer Cartridge

This example includes exemplary specifications used to design andassemble a microfluidic cartridge as well as exemplary instructions onthe use of the cartridge in, for example, the apparatus describedelsewhere herein.

Characteristics

In some embodiments, the cartridge has the following functionalspecifications.

The cartridge includes fluidic components (e.g., microchannels, valves,end vents, reagent inlet holes, reaction chambers, and the like)necessary to perform the functions of the cartridge (e.g., PCR).

The cartridge can be adapted to a one-time use, making it a disposablecartridge that can be disposed of according to typical laboratoryprocedures.

The cartridge is 4.375 inches long and 2.800 inches wide, with athickness of 0.094+/−0.005 inches. The cartridge includes features thatallow it to interface with, for example, the system described herein.The interfacing features include PCR channel walls and the top of themicro-substrate over the PCR channel that are well polished (SPIA1/A2/A3), enabling easy transfer of excitation and emission lightbetween the PCR reactor (contained in the cartridge) and a detectionsystem. The cartridge includes a thermal interface, located on thebottom of the cartridge, for interfacing with the analyzer. The thermalinterface includes a thin laminate (typically less than 150 micronsthick, such as 100 microns thick) to encourage heat transfer from theheater wafer to, for example, the PCR channels of the cartridge.

The cartridge includes one or more mechanical interfaces with, forexample, the receiving bay of a diagnostic apparatus as describedelsewhere herein. For example, the cartridge has a notch in one or moreof the corners that can mate with a corresponding shape on the heatermodule of the receiving bay. The notch and corresponding shape enablesthe cartridge to be placed only one way in the receiving bay. In someembodiments, the cartridge has a single notch in one of the corners,with the remaining three corners having a minimum radius of 1 mm tofacilitate placement of the cartridge in the analyzer.

The cartridge includes a number of inlet holes that are cone-shaped witha height of 1 mm from the top surface of the cartridge. The cone has aninner diameter of 3 mm at the top of the cone and tapers uniformly downto a diameter that matches the width of a microchannel (e.g., an inletchannel) to which the inlet cone is fluidly connected. The inlet channelfluidly connects the inlet hole to a PCR reactor that has an interiorvolume of, for example, about 4.25 μl to 4.75 μl (e.g., 4.22 μl, 4.5 μl,4.75 μl, or the like). An outlet microfluidic channel fluidly connectsthe PCR reactor to an overflow chamber. The cartridge also includes anoutlet vent hole.

The microfluidic substrate layer of the cartridge can include one ormore of the following specifications. The material of the microsubstrateis optically clear (e.g., has about 90% or greater optical transmission,be 3 mm thick, comply with ASTMD1003, and the like), haveauto-fluorescence that is less than that emitted by 2 mm thick ZEONOR1420R, and have a refractive index of about 1.53 (ASTM D542). Thematerial of the microsubstrate is amenable to the injection molding offeatures required for the microfluidic networks of the cartridge. Thematerial is typically compatible with all PCR agents and can withstandtemperatures of up to about 130° C. for about 5 minutes or more withoutyielding or melting.

The cartridge includes fiducials, recognizable by suitably configuredmanufacturing equipment, located in one or more (preferably two) of thecorners of the substrate.

Additional features of the substrate material include one or more of thefollowing. Minimum clearances of about 1 mm are present between adjacentfunctional features to ensure sealing success (e.g., to the analyzer),and to allow simplified fixturing during assembly. The cartridge caninclude “dogbones” under small fluid path ends to, for example, increasemold life. The bottom surface of the cartridge can be roughened (e.g.,by vapor hone, EDM, or the like prior to attaching laminate to thesubstrate). The substrate material is capable of adhesion by a label.

In some embodiments, the sealing tape used in the cartridge includes oneor more of the following specifications. Laminate can be easily appliedto the bottom of the microfluidic substrate. The material of thelaminate is essentially pin-hole free. The material of the sealing tape,in particular the adhesive, are compatible with the PCR reactionchemistries. The laminate material and glue used should notauto-fluoresce. The material can withstand up to 130° C. for 5 minuteswithout losing adhesion, and without yielding, melting, or causing unduestresses on the cartridge. Bubbles should not form in the adhesive layerupon heating (e.g., to 130° C. for 5 minutes) after application to themicrosubstrate. The laminate is less than 5 mils thick (typically 4 milsthick) to, for example, enable rapid heat transfer. (1 mil= 1/1000inch=25.4 microns.)

The high temperature wax included in the cartridge has the followingcharacteristics. The wax should have a melt point of about 90+/−3° C.(e.g., 87° C., 90° C., 93.1° C., or the like), be biocompatible with PCRreactions, have wettability with microsubstrate material, and have amelt viscosity range, for example, of about viscosity at 100° C.=20mm²/s and Hardness at 25° C.=8 dmm.

The main label on the upper surface of the cartridge has the followingcharacteristics. It has a thickness of 2-4 mils, has suitablebondability to micro features, and seals around the valves, includescuts for one or more PCR windows, and optionally space for a tab (freefrom adhesive) for aiding in removal of the cartridge from the analyzer.In embodiments, where a lid of a receiving bay for the cartridge slidesover the cartridge, the main label also has resistance to abrasion onits upper surface, and is printable. The main label can have an upperand lower alignment pattern for the label to completely cover the valveholes to permit proper operation of the valves.

The cartridge includes a barcode label applied to the top of thecartridge, which is readable by a barcode reader (e.g., the barcodereader included in the apparatus described elsewhere herein) while thecartridge is installed in the analyzer. The barcode label can includethe product name, lot #, expiration date, bar code (2D) and may beprinted directly onto the cartridge. In addition, or in the alternative,a barcode may be applied directly to the main cartridge label using alaser or inkjet type printer. The label is suitable for being read by ascanning head, for example as depicted in FIG. 30.

The packaging in which the cartridge is shipped includes one or more ofthe following: package label, carton, carton label, and/or operatinginstructions. The packaging can be printed on or can have a labelattached to it. The cartridge can be placed inside of a plastic bag,shrink/stretch wrap bag, or the like. The cartridge can be stacked andpackaged in groups, such as of 12, or 24. It is not critical that thecartridge bagging has a seal but it should be kept free fromcontamination, such as dust.

Microfluidic Components

The cartridge includes microchannels and holes such that the holes areof a size and shape to enable easy, leak-free interfacing with a 175 μlpipette tip. In some examples, the hole size is between about 200 μm andabout 4000 μm in diameter. The microchannels can be between about 50 μmand about 1500 μm wide and between about 50 μm and 1000 μm high.

The cartridge includes valves for controlling the flow of fluid withinthe cartridge (e.g., through the microchannels, reactor chambers, andthe like). The valve edges, steps, and general geometry can be designedto encourage exact flow and/or stoppage required during wax load. Thevalve geometry can be designed to accommodate limitations of waxdispensing equipment (e.g., +/−25% of 75 nL volume). In someembodiments, step down air chambers on the valves are funnel shaped toaid wax loading and the remaining geometry diminishes from the bottom ofthe funnel to the end point where the wax stops. The path where thevalves are to flow into and block, during use, is narrow enough (e.g.,150-200 microns wide and deep), and has enough length, to effectivelyseal when the valves are activated during use. The valve wax temperaturecan be about 90° C. When in use to block a portion of a microchannel,the valves seal to prevent evaporation of fluid and/or physicalmigration of fluid from the PCR reactor during thermocycling.

The cartridge includes one or more valves (such as temperaturecontrolled, wax-containing valves) for starting, stopping, and/orcontrolling the flow of material inside the cartridge. The wax containedin the valves is free of trapped air bubbles that have a diametergreater than half the width of the valve channel. The valve channel canhave an air pocket. The wax may not intrude into the fluid path (the150μ channel) prior to activation. The wax can be filled to the start ofthe flare at the fluid path (i.e., the junction of the 150μ fluidchannel and the wax loading channel, see, e.g., FIG. 9A).

The cartridge can include one or more PCR regions for performing PCR ona sample. The channel in the PCR region (the PCR reactor) can bedesigned such that the temperature of the contents of the channel remainuniformly within about 1° C. of the PCR anneal temperature. The channelwalls can have a polish of SPI A1/A2/A3.

Operation

An input PCR sample (e.g., a reaction mixture) can be between about 6.0and 7.0 μl per PCR lane (e.g., 5.9 μl per lane, 6.4 μl per lane, 7.1 μlper lane, or the like) and is introduced into the cartridge through theinlet hole by, for example, a pipette. A reaction mixture istransported, via the inlet channel, to the PCR reactor where thereaction mixture is isolated (e.g., sealed off by valves) to preventevaporation or movement (leakage) of the reaction mixture duringthermocycling. Once the various reaction mixtures are sealed insidetheir respective chambers, the diagnostic apparatus initiatesmultiplexed real-time PCR on some or all of the reaction mixtures (e.g.,4.5 μl, an amount of fluid equal to the inner volume of the reactionchamber, or the like).

In some embodiments, the cartridge is designed to be able to performdiagnostic tests within a temperature range of about 59° F. to about 86°F. (about 15° C. to about 30° C.) and a humidity range of about 15%relative humidity to about 80% relative humidity. The cartridge isdesigned to be safe and functional when used indoors, used at analtitude of 2000 m or less, and used under non-condensing humidityconditions (e.g., maximum relative humidity of 80% for temperatures upto 31° C. decreasing linearly to 50% relative humidity at 40° C.).

In use, end-product of PCR, produced in the cartridge, can remain in theused cartridge to, for example, minimize the likelihood of crosscontamination.

The cartridge can be designed such that a drop from a height of 4 foot,while in its packaging, will not damage the cartridge. The cartridge isdesigned to perform without damage after exposure to the followingconditions. The cartridge should be stored at 4° C. to 40° C. for therated shelf life. Exposure to temperatures between −20° C. and 4° C. or40° C. and 60° C. should occur for no longer than 24 hours. Thecartridge can withstand air pressure changes typical of air transport.

The cartridge can be labeled with the following information (e.g., toidentify the cartridge, comply with regulations, and the like). Thelabel can contain a “Research Use Only” label, if applicable, and a CEmark, if applicable. The label can contain the company name and logo(e.g., HandyLab®), a part number (e.g., 55000009), a part name (12xCartridge-nonvented), a lot number (e.g., LOT 123456), an expirationdate (e.g., in month and year format, such as “06/2015”), space forwriting, a barcode according to barcode specifications (describedelsewhere), and/or manufacturer name and address, e.g., “Handylab, Inc.,Ann Arbor, Mich. 48108 USA”.

The cartridge can be provided in a carton that contains information onits exterior or interior such as: a part number (e.g., 55000009), a partname (12x Cartridge-nonvented), a quantity (e.g., 24), a lot number(e.g., LOT 123456), an expiration date (e.g., 06/2015), an optional UPCcode, a manufacturer's statement and contact details, such as“Manufactured by Handylab, Inc., Ann Arbor, Mich. 48108 USA”, a cartonlabel to state storage limits, a CE mark (if applicable), and/or an ARname and address.

The cartridge packaging can include paper wrap to secure multiplecartridges together, and clean package fill to prevent damage, forexample, from vibration. The cartridge shipping carton can includefeatures such as, compliance to ASTM 6159, carton may be stored in anydirection, refrigeration or fragile labeling of the carton may not berequired, and additional cold packs may not be required. The shelf lifeof the cartridge is 12 months or more.

The cartridge complies with IEC 61010 (NRTL tested) and an FDA listingmay be required for clinical distribution. Cartridges used in a clinicallab device typically meet all quality system requirements. Cartridgesused for research only in a commercial device may meet all qualitysystem requirements of a particular research institution, or HandyLab,Inc. Cartridges for research use only (Alpha or Beta testing) may bedesign/manufacturing traceable to a device history record (DHR) (such asa manufacturing record).

The cartridge has a maximum limit of detection equal to 20 copies perreaction volume (i.e., 20 copies/4μ), with a target detection of 10copies per reaction volume. The cartridge can perform 45 reaction cyclesin 40 minutes or less (e.g., 45 cycles in 40 minutes, 45 cycles in 20minutes, 45 cycles in 15 minutes). The cartridge can utilize two colordetection using, for example, the FAM (or equivalent) and CAL RED (orequivalent) fluorescent dyes. Results obtained using the cartridge havebeen compared with the results obtained using standard real-time PCRinstruments.

During use (e.g., when placed in a system described herein andperforming a function such as PCR), the cartridge is pressed, on oneside, by the optics block, against the heater wafer (positioned againstthe opposite side), with a pressure of about 1 psi or greater (e.g.,0.99 psi, 1.2 psi, or the like). When located in the tray of theanalyzer, the cartridge has an alignment slop of +/−200 microns toenable a user to easily place and remove the cartridge from the analyzertray. The cartridge has two ledges, that are each 1 mm wide and locatedalong the two long edges of the cartridge, to enable the heating (lower)surface of the cartridge to extend below the datum of the tray andtherefore make effective contact with the upper surface of the heaterunit.

Cartridge Manufacturing:

Existing semi-automatic equipment for laminating & waxing (Think &Tinker DF-4200, & Asymtek Axiom Heated Jet Platform, respectively) canbe utilized to meet all cartridge manufacture requirements. An exemplaryprotocol is as follows:

-   -   Laminate micro substrate & trim excess.    -   Fill valves with hot wax & inspect.    -   Apply label & barcode.    -   Band 24 pieces together.    -   Bag & seal banded cartridges, label bag.    -   Place bag & insert(s) into carton, seal and label.

Venting is not required on this cartridge, which eliminates the mosttime consuming process for cartridge manufacture, along with the highestrisk and highest cost for fully integrated automation. (In embodiments,where a hydrophobic membrane needs to be attached, the membrane requirescareful placement, alignment, and pressure sealing at a properlyregulated temperature.) Over 1,000 pieces of the 12-up without ventinghave been successfully produced.

Example 2 Exemplary Chemistry and Processes of Use

The chemistry typically performed in conjunction with the microfluidiccartridge herein centers around the detection and identification oforganisms in a clinical specimen, by virtue of detecting nucleic acidsfrom the organism in question. This involves isolation of nucleic acidsfrom target organisms that are contained in a clinical specimen,followed by a process that will detect the presence of specific nucleicacid sequences. In addition to target detection, an internal positivecontrol nucleic acid is added to the collection buffer, and is takenthrough the entire extraction and detection process along with targetnucleic acids. This control monitors the effectiveness of the entireprocess and minimizes the risk of having false negative results.

Nucleic Acid Extraction and Purification

Nucleic acid extraction procedures begin with the addition of a clinicalspecimen to a prepared specimen collection solution. This can be doneeither at a specimen collection site, or at the testing site. Twocollection solution formats are available: one for body fluids, and onefor swab specimens. Collection solutions used at collection sites serveas specimen transport solutions, and therefore, this solution mustmaintain specimen and analyte integrity.

The extraction and purification procedure, which can be entirelyautomated, proceeds as follows:

-   -   Target organisms are lysed by heating the detergent-containing        collection solution.    -   Magnetic beads, added to the specimen/collection solution mix,        non-specifically bind all DNA that is released into the        solution.    -   Magnetic beads are isolated and are washed to eliminate        contaminants    -   DNA is released from the beads using high pH and heat.    -   DNA containing solution is removed and neutralized with a buffer        Nucleic Acid Amplification

Nucleic acids that have been captured by magnetic beads, washed,released in high pH, and neutralized with buffer, are added to a mixtureof buffers, salts, and enzymes that have been lyophilized in a tube. Themixture is rapidly rehydrated, and then a portion of the solution isloaded onto a microfluidic cartridge. The cartridge is then loaded intothe amplification instrument module, which consists of a heating unitcapable of thermal cycling, and an optical detection system. Detectionof target nucleic acids proceeds as follows:

-   -   The liquid is sealed in a reaction chamber.    -   Rapid thermal cycling is used to potentiate the Polymerase Chain        Reaction (PCR), which is used to amplify specific target DNA.    -   Amplified DNA fluoresces, and can be detected by optical        sensors.    -   A fluorescent probe “tail” is incorporated into each amplified        piece of DNA    -   At a specific temperature, the probe adopts a conformation that        produces fluorescence (this is termed a “scorpion” reaction).    -   Fluorescence is detected and monitored throughout the reaction.        Extraction and Amplification/Detection Process

Extensive bench-scale testing has been carried out to optimize thenucleic acid extraction chemistry, including the collection buffer, thewash buffer formulation, the release solution formulation, and the PCRreagent mixes. The fully automated method of extraction, followed by12-up PCR, was able to provide very high sensitivity consistently at 150copies/sample.

Examples: Chlamydia in Urine (50/50); Gonorrhoea in Urine; GBS inPlasma.

Various detection chemistries, such as Taqman, Scorpion, SYBRg Green,work reliably in the microfluidic cartridge.

Reagent Manufacturing

Feasibility studies were conducted in order to determine whether PCRreagents could be lyophilized in PCR tubes besides the use of 2 μllyophilized pellets. The studies have indicated that sensitivity ofreactions performed using tube-lyophilized reagents is equivalent tothat of wet reagents or 2 μl pellet reagents, so feasibility has beenproven. Stability studies for this format indicate similar stabilitydata. 2 microliter lyophilized PCR pellets have been found to be stableto up to 2 years at room temperature, once sealed in nitrogenatmosphere.

There are currently seven individual, blended chemistry componentsidentified for potential use with the system described herein. Mixing,blending and processing reagents/chemicals can be performed at HandyLab,Inc., with existing equipment already in place.

Collection buffer, wash, release and neutralization liquids are simplerecipes with very low risk, and can be made in large batches to keeplabor costs of mixing/blending at or below targeted projections. Theycan be mixed and placed into intermediate containers for stock, and thenissued to a manufacturer of reagent holders for dispensing.

Affinity Beads (AB) have good potential to be stored and used as aliquid in the strip, but design contingencies for using a lyophilizedpellet are in place as a back up. It is important to keep the beadssuspended in solution during dispense. Dispense equipment (e.g.,manufactured by Innovadyne) that provides agitation for continuoussuspension during dispense has been identified for achieving this, oncestability has been proven for liquid AB storage in the strip. Theprocess to manufacture and magnetize the Affinity Beads spans a 9 hourcycle time to produce a batch of 2,000 aliquots, but that same timeperiod can be used for scaled up recipe batches once we ramp into highvolume production. This item has the highest labor content of allchemistry manufacture that is currently required for the apparatus.

PCR reagents/enzymes can be freeze-dried in a lyophilizing chamber(e.g., Virtis Genesis) but will not require spherical pellet formation.Instead, the mixture can be dispensed into, and then lyophilized, insidethe end-use tube. First the various reagents are mixed, and then thefollowing steps are performed to accomplish lyophilization: Individualtubes are placed into a rack/fixture, and the solution is dispensed intoeach, using existing equipment (e.g., EFD Ultra Dispense Station). Thefilled rack is placed inside a stainless steel airtight box (modified toaccept stoppers in the lid) and then placed into the lyophilizationchamber, and the drying cycle commences unattended. Duringlyophilization, the stoppers are in a raised position allowingair/nitrogen to circulate into, and moisture to exit the stainless boxholding racks of vials. At the end of the cycle, the shelves of thelyophilization chamber lower to seat the stoppers into the lid, forminga seal while still inside the closed chamber, in a moisture freenitrogen atmosphere. The steel boxes are then removed from the chamber,and each rack inside is processed in a single operation to seal allvials in that rack. Immediately after sealing, the vials are die cutfrom the foil in one operation, allowing individual vials to beforwarded to the manufacturer for placement into a reagent holder.Internal control can either be added to an existing solution, or can bedispensed into its own cavity in the manner of the collection buffer,wash, neutralization, and release solutions. If lyophilization isrequired, it will be accomplished in the same manner as the PCRchemistry, and later snapped into the strip. Shelf life stabilitystudies are underway.

Real-Time PCR

After all the appropriate PCR lanes of the PCR cartridge are loaded withfinal PCR solution, the tray containing the cartridge moves it in thePCR Analyzer. The cartridge is pressed by the optical detectionread-head against the heater. Heaters activate valves to close eitherends of the PCR reactor and real-time thermocycling process starts.After completing appropriate PCR cycles (˜45 cycles), the analyzerdecides whether the sample has the target DNA based on the outputfluorescence data.

Example 3 Exemplary Diagnostic Apparatus

The apparatus, in combination with the associated consumables (reagents,cartridges, etc.), automatically performs all aspects of nucleic acidtesting, including sample preparation, amplification, and detection forup to 48 samples per hour with the first 24 results available in lessthan an hour. The system is straightforward to use. An operator simplyaliquots a portion of the patient sample into a dedicated tube thatcontains pre-packaged buffer. The operator places the dedicated tubesinto positions on a sample rack. The operator then loads a disposableplastic reagent strip for the appropriate test in the rack. The onlyother consumable used in the apparatus are microfluidic PCR cartridgesfor conducting amplification and detection; each cartridge is capable ofperforming up to twelve PCR tests and two cartridges can be loaded intothe analyzer at once. Should the apparatus require a new PCR cartridge,the apparatus will prompt the operator to load the cartridge. Theapparatus will then prompt the operator to close the lid to initiatetesting. All consumables and sample tubes are barcoded for positivesample identification.

Sample lysis and DNA preparation, which will require approximately halfan hour for a full run of 24 samples, is automatically performed by theanalyzer's robotic and liquid handling components using protocols andreagents located in unitized, disposable plastic strips. The apparatusthen automatically mixes the sample and PCR reagents, and injects themixture into a cartridge that will be automatically processed by anintegrated PCR machine. Rapid, real time PCR and detection requires lessthan 20 minutes. Results, which will be automatically available uponcompletion of PCR, are displayed on the instrument's touch screen,printed or sent to the hospital information system, as specified by theuser (or the user's supervisor).

Each instrument can process up to 24 samples at a time with a totalthroughput of 48 samples per hour after the first run. The apparatus isslightly less than 1 m wide and fits easily on a standard lab bench. Alloperations of the unit can be directed using the included barcode wandand touch screen. The analyzer can be interfaced with lab informationsystems, hospital networks, PCs, printers or keyboards through four USBinterfaces and an Ethernet port.

The apparatus has the following characteristics.

Sensitivity: the apparatus will have a limit of detection of ˜50 copiesof DNA or RNA. (and may have a limit of detection as low as 25-30 copiesof DNA/RNA).

Cost per Test: Due to the miniaturized, simplified nature of thereagents described herein and also in U.S. patent application Ser. No.12/172,208, filed Jul. 11, 2008 and incorporated herein by reference,cartridge, and other consumables, the cost of goods per test will berelatively low and very competitive.

Automation: By contrast with current “automated” NAT systems, which allrequire some degree of reasonably extensive technologist interactionwith the system, through the use of unitized tests and full integrationof sample extraction, preparation, amplification and detection, theapparatus herein will offer a higher level of automation, andcorresponding reduction in technologist time and required skill level,thereby favorably impacting overall labor costs.

Throughput: Throughput is defined as how many tests a system can conductin a given amount of time. The apparatus will be capable of running 45tests per hour, on average.

Time to First Result: In a hospital environment, time to first result isan especially important consideration. The apparatus will produce thefirst 24 results in less than an hour and an additional 24 results everyhalf hour thereafter.

Random Access and STAT: Random access is the ability to run a variety oftests together in a single run and place samples in unassigned locationson the analyzer. Also, with chemistry and immunoassay systems, it isdesirable to be able to add tests after a run has started. This is oftenreferred to as “true random access” since the user is provided completeflexibility with regard to what tests can be run where on an analyzerand when a new sample can be added to a run. A STAT is a sample thatrequires as rapid a result as possible, and therefore is given priorityin the testing cue on the analyzer. Today, essentially all chemistry andimmunoassay analyzers are true random access and offer STATcapabilities. For NAT, however, very few systems offer any random accessor STAT capabilities. The instrument herein will provide random accessand STAT capabilities.

Menu: The number and type of tests available for the analyzer is a veryimportant factor in choosing systems. The apparatus herein deploys alaunch menu strategy that involves a mix of high volume, “standard”nucleic acid tests combined with novel, high value tests.

The apparatus enables 24 clinical samples to be automatically processedto purify nucleic acid, mix the purified DNA/RNA with PCR reagents andperform real-time PCR in microfluidic cartridge to provide sample toresults in an hour. The exemplary apparatus has two PCR readers, eachcapable of running a 12 lane microfluidic cartridge using an opticalsystem that has dedicated two-color optical detection system.

The apparatus has the following sub-systems:

-   -   Two sample processing racks, each rack processes up to 12        clinical samples in unitized disposable strips    -   Magnetic separator-cum-tube heater assembly (24 heating        stations)    -   A four-probe liquid dispensing head    -   3-axis gantry to move the pipette head    -   Two PCR amplification-detection stations, each capable of        running PCR in the lanes of a 12-lane microfluidic cartridge,        and dedicated 2-color optical detection system for each PCR        lane.    -   Control electronics    -   Barcode reader

Operation: The user gets a work list for each sample, whether they wantto detect certain target analyte (such as GBS, Chlamydia, Gonorrhoea,HSV) for each clinical sample. The sample tubes are placed on the rackand for each sample, the user slides in a disposable reagent holder(analyte specific) into a corresponding lane of the rack. The reagentholder has all the sample prep reagents, PCR reagents, process tubes aswell as disposable pipettes already prepackaged in it. Once alldisposables are loaded into the rack, the rack is placed in its locationon the instrument. The user then places two 12-lane microfluidic PCRcartridges in the two trays of the PCR reader. The user then closes thedoor of the instrument and starts the sample processing using the GUI(Graphical User Interface).

The instrument checks functionality of all subsystems and then reads thebarcode of the sample tubes, the unitized reagent disposables and themicrofluidic cartridges. Any mismatch with a pre-existing work list isdetermined and errors are flagged, if necessary. The instrument thangoes through a series of liquid processing, heating, magnetic separationto complete the sample preparation steps for the each of the clinicalsample, mixes the purified nucleic acid with PCR reagents and dispensesthe final mix into a lane of the microfluidic cartridges. After amicrofluidic cartridge is loaded with the final PCR mix, the cartridgetray moves and aligns the cartridge in the reader and the opticaldetection system presses the cartridge against a microfluidic PCR heatersurface. On-chip valves are actuated to close the reaction mix and thenthermocycling is started to initiate the PCR reaction. At each cycle ofPCR (up to 45 cycles), fluorescence from each PCR lane is detected bythe optical detection system (2-colors per PCR lane) and final result isdetermined based on the threshold cycle (Ct).

The sample preparation steps for 24 samples are performed in about 40minutes and the PCR reaction in about 20 minutes.

Sample Reader

The Reader performs function testing of up to twelve properly preparedpatient samples by PCR process (real-time PCR) when used in conjunctionwith microfluidic (test) cartridges as further described herein. Eachunit employs two Reader Modules for a total of up to twenty four tests.(FIGS. 31A and 31B) Operation of the Reader is designed for minimal userinteraction, requiring the loading and unloading of test cartridgesonly. During the “Load Disposables” sequence, the Reader will present amotor actuated tray for installation of the disposable cartridge.Sliding a small knob located in the front of the tray, a spring loadedprotective cover will raise allowing the test cartridge to be nestedproperly in place. The cover is then lowered until the knob self-locksinto the tray frame, securing the cartridge and preventing movementduring the sample loading sequence.

Once the prepared samples have been dispensed via pipettes into thecartridge, the tray will retract into the Reader, accurately positioningthe test cartridge beneath the chassis of the optical assembly. Theoptical assembly will then be lowered by a captured screw driven steppermotor until contact is made with the test cartridge. At this point thetest cartridge is located ⅛″ above the target location on the heaterassembly. As downward motion continues the test cartridge and its holderwithin the tray compress springs on the tray frame (these are used laterto return the cartridge to it's normal position and able to clear theencapsulated wire bonds located on the heater assembly during trayoperation). Movement of the test cartridge and optical assembly iscomplete once contact with the heater assembly is made and a minimum of2 psi is obtained across the two-thirds of the cartridge area about thePCR channels and their controlling gates. At this point the testing ofthe cartridge is performed using the heater assembly, measured withonboard optics, and controlled via software and electronics much in thesame manner as currently operated on similar diagnostic instruments.

Once the functional testing is complete the main motor raises the opticassembly, releasing pressure on the test cartridge to return to itsnormal position. When commanded, the tray motor operating in arack-and-pinion manner, presents the tray to the customer for cartridgeremoval and disposal. When the tray is in the extended position it issuspended above a support block located on the apparatus chassis. Thisblock prevents the cartridge from sliding trough the holder in the trayduring loading and acts as a support while samples are pipetted into thedisposable cartridge. Also provided in this support block is an assistlever to lift and grasp the disposable cartridge during removal. Allcomponents of the tray as well as support block and cartridge liftassist are removable by the customer, without tools, for cleaning andreinstalled easily.

Microfluidic PCR Heater Module

The microfluidic PCR heater module comprises a glass wafer withphotolithographically defined microheaters and sensors to accuratelyprovide heat for actuation of valves and performing thermocyclingrequired to perform a real-time PCR reaction. The wafer surface hasdedicated individually controlled heating zones for each of the PCRlanes in the microfluidic cartridge. For a 12-lane cartridge, there are12 PCR zones; in the 24-up cartridge, there are 24 PCR heating zones.The individual heaters and sensors are electrically connected to aPrinted circuit board using gold or aluminum wire bonds. A thermallycompliant encapsulant provides physical protection to the wirebonds.While the present device is made on glass wafer, heaters can befabricated on Si-on-Glass wafers and other polymeric substrates. Eachsubstrate can provide specific advantages related to its thermal andmechanical properties. Besides using photolithography process, suchheating substrates can also be assembled using off-the-shelf electroniccomponents such as power resistors, peltiers, transistors, maintainingthe upper heating surface of each of the component to be at the samelevel to provide heating to a microfluidic cartridge. Temperaturecalibration values for each temperature sensor may be stored in EEPROMor other memory devices co-located in the heater PC Board.

Example 4 Exemplary High-Efficiency Diagnostic Apparatus

A more highly multiplexed embodiment, enables 24 clinical samples to beautomatically processed to purify nucleic acids, mix the purifiedDNA/RNA with PCR reagents and perform real-time PCR in a microfluidiccartridge. This product has a single PCR reader, with a scanningread-head, capable of reading up to 4 different colors from each of thePCR lanes in a microfluidic cartridge. The cartridge has 24 PCR channelsenabling a single cartridge to run all 24 clinical samples. In addition,this apparatus includes a cartridge autoloader, whereby the instrumentautomatically feeds the PCR reader from a pack of cartridges anddiscards each used cartridge into a waste tray.

The apparatus has the same sub-systems as the apparatus of Example 3,herein, except in the following: a single PCR amplification-detectionstation capable of running a 24-lane microfluidic cartridge is used inplace of two such stations each running 12-lane cartridges; a scannerunit can detect up to 4 colors from each PCR lane in place of a 2-coloroptical detection system; and an autoloader unit to feed 24-lanemicrofluidic cartridge from a box into the PCR detection unit can beused in place of manual loading of a cartridge.

Operation of the instrument is similar to that of the instrument inExample 3, herein, except that a single 24-lane cartridge is loaded,either manually, or from the auto-loader and used cartridge is thenpushed out automatically into a waste cartridge bin. Microfluidiccartridges are stored in a cartridge pack (maximum 24 cartridges) andthe instrument alerts the user to replace the cartridge pack and emptyout the waste cartridge bin once all cartridges from the pack are usedup.

Example 5 Exemplary 24-Lane Cartridge

Various views of an exemplary 24-lane cartridge 5000 are shown in FIGS.32A (perspective view), 32B (plan view), and 32C (exploded view).

As shown in FIG. 32C, the cartridge 5000 has 3 layers, a laminate 5001,a substrate 5002, and a label 5003. The label is shown in two pieces inthis example. A barcode 5006 is visible on label 5003. A row of 24sample inlets 5004 is shown. A ledge 5005 runs along each of the longedges of the cartridge to facilitate placement in a removable receptaclein receiving bay of a diagnostic instrument, or in a cartridgeauto-loader, as further described elsewhere herein.

FIG. 32B has three plan views of the cartridge substrate (upper surface,edge, and lower surface), and two inset views. The upper inset view(“Detail A”) shows dimensions of a cut-out (notch) in the upper edge ofthe cartridge. The right inset view (“Detail B”) shows a pair ofadjacent lanes, each having a microfluidic network, and each from one ofthe two banks of networks. The chambers in adjacent networks (fromalternate banks) are staggered with respect to one another, even thoughthe sample inlets are all shown in a single line.

The 24-lane cartridge has two banks of 12 PCR lanes, shown in plan viewin FIG. 32B. Each lane has a liquid inlet port, that interfaces with adisposable pipette tip; a 4 microliter PCR reaction chamber labeled 3Ain the inset in FIG. 32B (1.5 mm wide, 300 microns deep, andapproximately 10 mm long), two microvalves on either side of the PCRreactor (one of which labeled 3B in the inset in FIG. 32B, and an outletvent. Microvalves are normally open initially and close the channel onactuation. The outlet holes enable extra liquid (˜1 μl) to be containedin the fluidic channel in case more than 6 μl of fluid is dispensed intoa lane of the cartridge.

Dimensions of the cartridge (in inches) and layout of the lanes in thecartridge are shown in FIG. 32B. It would be understood that thesedimensions and layout are exemplary, and deviations from those shown areconsistent with an equivalent manner of operation of such a cartridge.

The inlet holes 5004 of the cartridge are made conical in shape and havea diameter of 3-6 mm at the top to ensure pipettes can be easily landedby the fluid dispensing head within the conical hole. The bigger theholes, the better is the alignment with the pipette, however, there is atrade-off between maximizing the number of inlet ports within the widthof the cartridge and ensuring that the pitch between the holes iscompatible with the inter-pipette distance. In this particular design,the inter-pipette distance is 24 mm and the distance between the loadingholes in the cartridge is 8 mm. So lanes 1, 4, 7, 11 are pipetted intoduring one dispensing operation; lanes 2, 5, 8 and 12 in the next, andso on and so forth.

The height of the conical holes is kept lower than the height of theledges on the sides of the cartridge to ensure the cartridges can bestacked on the ledges. The ledges on the two long edges of the cartridgeenable stacking of the cartridges with minimal surface contact betweentwo stacked cartridges and also help guide the cartridge into the readerfrom a cartridge pack in an auto-loader.

The foregoing description is intended to illustrate various aspects ofthe present inventions. It is not intended that the examples presentedherein limit the scope of the present inventions. The technology nowbeing fully described, it will be apparent to one of ordinary skill inthe art that many changes and modifications can be made thereto withoutdeparting from the spirit or scope of the appended claims.

1. A microfluidic substrate, comprising: a plurality of sample lanes,wherein each of the plurality of sample lanes comprises a microfluidicnetwork having, in fluid communication with one another: an inlet; afirst valve and a second valve; a reaction chamber; a vent; a firstchannel leading from the inlet, via the first valve, to the reactionchamber; and a second channel leading from the reaction chamber, via thesecond valve, to the vent; wherein the first and second valves areco-planar with the first and second channels in the microfluidic networkand wherein the first and second valves each comprise a temperatureresponsive substance that melts upon heating and seals the first andsecond channels.
 2. The microfluidic substrate of claim 1, wherein eachof the plurality of sample lanes is configured to amplify one or morepolynucleotides independently of the other lanes.
 3. The microfluidicsubstrate of claim 1, configured to carry out real-time PCR in at leastone of the reaction chambers.
 4. The microfluidic substrate of claim 1,wherein each of the inlets is configured to accept a quantity of samplefrom a pipette tip.
 5. The microfluidic substrate of claim 4, whereinthe quantity of sample is from 1-20 μl.
 6. The microfluidic substrate ofclaim 4, wherein an inlet comprises an inverted frustoconical structureof at least 1 mm height, and having a diameter at its widest point thataccepts entry of a pipette tip, of from 1-5 mm.
 7. The microfluidicsubstrate of claim 1, wherein the inlets of the respective plurality ofsample lanes are spaced apart from one another to permit simultaneousloading from a multiple-pipette head dispenser.
 8. The microfluidicsubstrate of claim 1, wherein the reaction chamber has a volume of 3-5μl.
 9. The microfluidic substrate of claim 1, wherein the plurality ofsample lanes is 12 lanes.
 10. The microfluidic substrate of claim 1,further comprising a fluorescence detection window situated over each ofthe reaction chambers.
 11. A microfluidic cartridge comprising themicrofluidic substrate of claim
 1. 12. The microfluidic cartridge ofclaim 11, further comprising a registration member that ensures that thecartridge is received by a complementary diagnostic apparatus in asingle orientation.
 13. The microfluidic cartridge of claim 11, whereineach of the microfluidic networks, including the reaction chambers, theinlet holes and the valves for isolating the reaction chambers, isdefined in a single substrate.
 14. The microfluidic cartridge of claim11, wherein the substrate is a rigid substrate and impervious to air orliquid, and entry or exit of air or liquid during operation of thecartridge is only possible through the inlet or a vent.
 15. Amicrofluidic cartridge, comprising: a substrate having an upper side andan opposed lower side, wherein the substrate comprises a plurality ofsample lanes, wherein each lane of the plurality of sample lanescomprises a microfluidic network having, in fluid communication with oneanother: an inlet; a first valve and a second valve; a first channelleading from the inlet, via the first valve, to a reaction chamber; asecond channel leading from the reaction chamber, via the second valve,to a vent; and a label, attached to the upper side, wherein each valvehas a wax loading hole extending to the upper side of the substrate, andwherein the label covers and seals the loading holes.
 16. The cartridgeof claim 15, wherein the microfluidic network is configured so that thetime required to fill from the inlet to the second valve is less than50% of the time required to fill up to the exit vent.
 17. The cartridgeof claim 15, wherein each microfluidic network comprises an overflowreservoir to contain extra liquid dispensed into the cartridge.
 18. Thecartridge of claim 15, wherein a 2-D barcode sample identifier ispresent on the label.
 19. The cartridge of claim 15, further comprisingtwo or more positioning elements for use when filling the valves withthermally responsive material.
 20. The cartridge of claim 15, configuredto be stackable.
 21. The cartridge of claim 15, wherein the substratecomprises two ridges, one of each situated along each of two oppositeedges of the cartridge, the ridges disposed on the upper side of thesubstrate.
 22. The cartridge of claim 21, wherein the two ridges areconfigured to permit the cartridge to be supported horizontally on atray, but keeping the lower side of the substrate below the tray.
 23. Amicrofluidic substrate, comprising: a plurality of sample lanes, whereineach of the plurality of sample lanes comprises a microfluidic networkhaving, in fluid communication with one another: an inlet; a first valveand a second valve; a reaction chamber; a vent; a first channel leadingfrom the inlet, via the first valve, to the reaction chamber; and asecond channel leading from the reaction chamber, via the second valve,to the vent; wherein the first and second valves comprise a temperatureresponsive substance that melts upon heating and seals the reactionchamber.
 24. The microfluidic substrate of claim 23, wherein each of theplurality of sample lanes is configured to amplify one or morepolynucleotides independently of the other lanes.
 25. The microfluidicsubstrate of claim 23, configured to carry out real-time PCR in at leastone of the reaction chambers.
 26. The microfluidic substrate of claim23, wherein each of the inlets is configured to seal with and accept aquantity of sample from a pipette tip.
 27. The microfluidic substrate ofclaim 26, wherein the quantity of sample is from 1-20 μl.
 28. Themicrofluidic substrate of claim 26, wherein an inlet comprises aninverted frustoconical structure of at least 1 mm height, and having adiameter at its widest point that accepts entry of a pipette tip, offrom 1-5 mm.
 29. The microfluidic substrate of claim 23, wherein theinlets of the respective plurality of sample lanes are spaced apart fromone another to permit simultaneous loading from a multiple-pipette headdispenser.
 30. The microfluidic substrate of claim 23, wherein thereaction chamber has a volume of 3-5 μl.
 31. The microfluidic substrateof claim 23, wherein the plurality of sample lanes is 12 lanes.
 32. Themicrofluidic substrate of claim 23, further comprising a fluorescencedetection window situated over each of the reaction chambers.
 33. Amicrofluidic cartridge comprising the microfluidic substrate of claim23.
 34. The microfluidic cartridge of claim 33, further comprising aregistration structure that ensures that the cartridge is received by acomplementary diagnostic apparatus in a single orientation.
 35. Themicrofluidic cartridge of claim 33, wherein each of the microfluidicnetworks, including the PCR reaction chambers, the inlet holes and thevalves for isolating the PCR reaction chambers, is defined in a singlesubstrate.
 36. The microfluidic cartridge of claim 33, wherein thesubstrate is a rigid substrate and impervious to air or liquid, andentry or exit of air or liquid during operation of the cartridge issubstantially possible only through the inlet or a vent.